Methods and pharmaceutical compositions for healing wounds

ABSTRACT

A pharmaceutical composition and method for inducing or accelerating a healing process of a skin wound are described. The pharmaceutical composition contains, as an active ingredient, a therapeutically effective amount of at least one agent for modulating PKC production and/or activation, and a pharmaceutically acceptable carrier. The method is effected by administering the composition to a wound.

[0001] This is a continuation-in-part of U.S. patent application Ser.No. 10/169,801, filed Jul. 9, 2002, which is a National Phase ofPCT/IL01/00675, filed Jul. 23, 2001, which claims priority of U.S.patent application Ser. No. 09/629,970, filed Jul. 31, 2000, nowabandoned. This application also claims the benefit of priority of U.S.provisional patent application No. 60/486,906, filed Jul. 15, 2003.

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention relates to a method and a pharmaceuticalcomposition for inducing and/or accelerating cell proliferation and/orcell differentiation and thereby accelerating the healing process ofwounds. More particularly, the present invention relates to the use ofmodulated expression and/or activation, e.g., as initiated by membranetranslocation, of serine/threonine protein kinases, also known as PKCs,for inducing and/or accelerating cell proliferation and/or celldifferentiation and thereby accelerating the healing process of wounds.Such modulated expression may be effected in accordance with theteachings of the present invention by (i) transformation of wound cellswith a PKC expressing construct; (ii) transformation of wound cells witha cis-acting element to be inserted adjacent to, and upstream of, anendogenous PKC gene of the wound cells; (iii) administration of insulinfor inducing expression and/or activation of PKC in wound cells; (iv)transformation of wound cells with an insulin expressing construct, whenexpressed and secreted the insulin produced therefrom serves as anup-regulator for PKC expression and/or activation; (v) transformation ofwound cells with a cis-acting element to be inserted adjacent to, andupstream of, the endogenous insulin gene of the wound cells, whenexpressed and secreted the insulin serves as an up-regulator for PKCexpression and/or activation; (vi) implantation of insulin secretingcells to the wound; (vii) transformation of wound cells with atrans-acting factor, e.g., PDX1, for induction of endogenous insulinproduction and secretion, the insulin serves as an up-regulator for PKCexpression and/or activation; and (viii) administration to the wound ofa PKC modulator.

[0003] The present invention, as is realized by any of the abovemethods, can also be practiced ex-vivo for generation of skin grafts.

[0004] The primary goal in the treatment of wounds is to achieve woundclosure. Open cutaneous wounds represent one major category of woundsand include burn wounds, neuropathic ulcers, pressure sores, venousstasis ulcers, and diabetic ulcers.

[0005] Open cutaneous wounds routinely heal by a process which comprisessix major components: (i) inflammation; (ii) fibroblast proliferation;(iii) blood vessel proliferation; (iv) connective tissue synthesis; (v)epithelialization; and (vi) wound contraction. Wound healing is impairedwhen these components, either individually or as a whole, do notfunction properly. Numerous factors can affect wound healing, includingmalnutrition, infection, pharmacological agents (e.g., actinomycin andsteroids), advanced age and diabetes [see Hunt and Goodson in CurrentSurgical Diagnosis & Treatment (Way; Appleton & Lange), pp. 86-98(1988)].

[0006] With respect to diabetes, diabetes mellitus is characterized byimpaired insulin signaling, elevated plasma glucose and a predispositionto develop chronic complications involving several distinctive tissues.Among all the chronic complications of diabetes mellitus, impaired woundhealing leading to foot ulceration is among the least well studied. Yetskin ulceration in diabetic patients takes a staggering personal andfinancial cost (29, 30). Moreover, foot ulcers and the subsequentamputation of a lower extremity are the most common causes ofhospitalization among diabetic patients (30-33). In diabetes, the woundhealing process is impaired and healed wounds are characterized bydiminished wound strength. The defect in tissue repair has been relatedto several factors including neuropathy, vascular disease and infection.However, other mechanisms whereby the diabetic state associated withabnormal insulin signaling impairs wound healing and alter thephysiology of skin has not been elucidated.

[0007] There is also a common problem of wound healing followingsurgical procedures in various parts of the body, the surgery succeedsbut the opening wound does not heal.

[0008] Skin is a stratified squamous epithelium in which cellsundergoing growth and differentiation are strictly compartmentalized. Inthe physiologic state, proliferation is confined to the basal cells thatadhere to the basement membrane. Differentiation is a spatial processwhere basal cells lose their adhesion to the basement membrane, ceaseDNA synthesis and undergo a series of morphological and biochemicalchanges. The ultimate maturation step is the production of the cornifiedlayer forming the protective barrier of the skin (1, 2). The earliestchanges observed when basal cells commit to differentiate is associatedwith the ability of the basal cells to detach and migrate away from thebasement membrane (3). Similar changes are associated with the woundhealing process where cells both migrate into the wound area andproliferative capacity is enhanced. These processes are mandatory forthe restructuring of the skin layers and induction of properdifferentiation of the epidermal layers.

[0009] The analysis of mechanisms regulating growth and differentiationof epidermal cells has been greatly facilitated by the development ofculture systems for mouse and human keratinocytes (2,4). In vitro,keratinocytes can be maintained as basal proliferating cells with a highgrowth rate. Furthermore, differentiation can be induced in vitrofollowing the maturation pattern in the epidermis in vivo. The earlyevents include loss of hemidesmosome components (3,5) and a selectiveloss of the α6β4 integrin and cell attachment to matrix proteins. Thissuggests that changes in integrin expression are early events inkeratinocyte differentiation. The early loss of hemidesmosomal contactleads to suprabasal migration of keratinocytes and is linked toinduction of Keratin 1 (K1) in cultured keratinocytes and in skin (1, 3,6). Further differentiation to the granular layer phenotype isassociated with down regulation of both β1 and β4 integrin expression,loss of adhesion potential to all matrix proteins and is followed bycornified envelope formation and cell death. Differentiating cellsultimately sloughs from the culture dish as mature squames (2, 7). Thisprogram of differentiation in vitro closely follows the maturationpattern of epidermis in vivo.

[0010] Recent studies in keratinocytes biology highlights thecontribution of Protein Kinase C pathways, which regulate skinproliferation and differentiation. The protein kinase C (PKC) family ofserine-threonine kinases plays an important regulatory role in a varietyof biological phenomena (8,9). The PKC family is composed of at least 12individual isoforms which belong to 3 distinct categories: (i)conventional isoforms (α, β1, β2, γ) activated by Ca²⁺, phorbol estersand diacylglycerol liberated intracellularly by phospholipase C; (ii)novel isoforms (δ, ε, η, θ) which are also activated by phorbol estersand diacylglycerol but not by Ca²⁺; and (iii) a typical (ζ, λ, ι)members of the family, which are not activated by Ca²⁺, phorbol estersor diacylglycerol.

[0011] On activation, most but not all isoforms are thought to betranslocated to the plasma membrane from the cytoplasm. The type ofisoform and pattern of distribution vary among different tissues and mayalso change as a function of phenotype. Numerous studies havecharacterized the structure and function of PKC because of itsimportance in a wide variety of cellular endpoints of hormone action.Five PKC isoforms—α, δ, ε, η and ζ—have been identified in skin in vivoand in culture. Recent studies have shown that the PKC signaltransduction pathway is a major intracellular mediator of thedifferentiation response (10,11). Furthermore, pharmacologicalactivators of PKC are powerful inducers of keratinocyte differentiationin vivo and in vitro (4, 12), and PKC inhibitors prevent expression ofdifferentiation markers (10).

[0012] While conceiving the present invention, it was hypothesized thatPKC isoforms over-expression and/or activation may be beneficial foraccelerating wound healing processes. The limitations for investigatingthe role of distinct PKC isoforms in skin cells proliferation and/ordifferentiation has been hampered as result of the difficulty inintroducing foreign genes efficiently into primary cells, byconventional methods. The short life span, differentiation potential andthe inability to isolate stable transformants do not allow efficienttransduction of foreign genes into primary skin cells.

[0013] Prior art describes the potential use of insulin as a therapeuticagent for healing wounds. Thus, U.S. Pat. Nos. 5,591,709, 5,461,030 and5,145,679 describe the topical application of insulin to a wound topromote wound healing. However, these patents describe the use ofinsulin in combination with glucose since the function of the insulin isto enhance glucose uptake and to thus promote wound healing.

[0014] U.S. patent application Ser. No. 09/748,466 and InternationalPatent Application No. PCT/US98/21794 describe compositions containinginsulin for topical application to skin for the purpose of improvingskin health or treating shallow skin injuries. However, none of thesepatent applications teaches the use of insulin for treating chronic,Grade II or deep wounds.

[0015] International Patent Application No. PCT/US01/10245 describes theuse of cyanoacrylate polymer sealant in combination with insulin orsilver for wound healing. However, the use of insulin in combinationwith another biologically active agent capable of modulating theexpression and/or activation of PKC is not taught nor suggested in thisapplication.

[0016] International Patent Application No. PCT/US85/00695 describestopical application of insulin for treating diabetes. However, thispatent application fails to teach the use of insulin for the purpose oftreating diabetes non-related wounds.

[0017] International Patent Application No. PCT/US92/03086 describestherapeutic microemulsion formulations which may contain insulin.However the use of the formulated insulin for the purpose of woundhealing is not taught in this disclosure.

[0018] U.S. Pat. Nos. 4,673,649 and 4,940,660 describe compositions forclonal growth of human keratinocytes and epidermal cells in vitro whichinclude epidermal growth factor and insulin. Both of these patents teachthe use of insulin for the development of cultured skin cells which maybe used for grafting. However, the application of insulin on wounds invivo is not taught by these patents.

[0019] None of the above cited prior art references teach or suggest theuse insulin for modulating the expression and/or activation of PKC, soas to accelerate the healing process of wounds. Furthermore, the priorart fails to teach or suggest utilizing nucleic acid constructs orgenetic transformation techniques for providing insulin to wounds, so asto accelerate the healing process of the wounds.

[0020] There is a widely recognized need for, and it would be highlyadvantageous to have, new approaches for accelerating the processesassociated with wound healing. In addition, there is a widely recognizedneed for, and it would be highly advantageous to have, an efficientmethod to insert recombinant genes into skin cells which will acceleratecell proliferation and/or differentiation processes and wound healing.

SUMMARY OF THE INVENTION

[0021] According to one aspect of the present invention there isprovided a method of inducing or accelerating a healing process of askin wound, the method comprising the step of administering to the skinwound a therapeutically effective amount of an agent for modulating PKCproduction and/or PKC activation.

[0022] According to another aspect of the present invention there isprovided a pharmaceutical composition for inducing or accelerating ahealing process of a skin wound, the pharmaceutical compositioncomprising, as an active ingredient, a therapeutically effective amountof at least one agent for modulating PKC production and/or activity; anda pharmaceutically acceptable carrier.

[0023] According to still another aspect of the present invention thereis provided a method of inducing or accelerating a healing process of askin wound, the method comprising the step of administering to the skinwound a therapeutically effective amount of insulin and at least oneadditional agent acting in synergy with the insulin, so as to induce oraccelerate the healing process of the skin wound.

[0024] According to yet another aspect of the present invention there isprovided a pharmaceutical composition for inducing or accelerating ahealing process of a skin wound, the pharmaceutical compositioncomprising, as an active ingredient, a therapeutically effective amountof insulin, at least one additional agent acting in synergy with theinsulin, and a pharmaceutically acceptable carrier being designed fortopical application of the pharmaceutical composition.

[0025] According to still another aspect of the present invention thereis provided a method of inducing or accelerating a healing process of askin wound, the method comprising the step of administering to the skinwound a single dose of a therapeutically effective amount of insulin,thereby inducing or accelerating the healing process of the skin wound.

[0026] According to an additional aspect of the present invention thereis provided a pharmaceutical composition for inducing or accelerating ahealing process of a skin wound, the pharmaceutical compositioncomprising, as an active ingredient, a single dose-unit of insulinselected capable of inducing or accelerating the healing process of theskin wound, and a pharmaceutically acceptable carrier being designed fortopical application of the pharmaceutical composition.

[0027] According to yet another aspect of the present invention there isprovided a method of inducing or accelerating a healing process of anold skin wound, the method comprising the step of administering to theold skin wound a single dose of a therapeutically effective amount ofinsulin, thereby inducing or accelerating the healing process of the oldskin wound.

[0028] According to still another aspect of the present invention thereis provided a method of inducing or accelerating a healing process of askin wound, the method comprising the step of implanting into the skinwound a therapeutically effective amount of insulin secreting cells, soas to induce or accelerate the healing process of the skin wound.

[0029] According to yet another aspect of the present invention there isprovided a pharmaceutical composition for inducing or accelerating ahealing process of a skin wound, the pharmaceutical compositioncomprising, as an active ingredient, insulin secreting cells, and apharmaceutically acceptable carrier being designed for topicalapplication of the pharmaceutical composition.

[0030] According to an additional aspect of the present invention thereis provided a method of inducing or accelerating a healing process of askin wound, the method comprising the step of transforming cells of theskin wound to produce and secrete insulin, so as to induce or acceleratethe healing process of the skin wound.

[0031] According to yet an additional aspect of the present inventionthere is provided a pharmaceutical composition for inducing oraccelerating a healing process of a skin wound, the pharmaceuticalcomposition comprising, as an active ingredient, a nucleic acidconstruct being designed for transforming cells of the skin wound toproduce and secrete insulin, and a pharmaceutically acceptable carrierbeing designed for topical application of the pharmaceuticalcomposition.

[0032] According to still an additional aspect of the present inventionthere is provided a method of inducing or accelerating a healing processof a skin wound, the method comprising the step of transforming cells ofthe skin wound to produce a protein kinase C, so as to induce oraccelerate the healing process of the skin wound

[0033] According to a further aspect of the present invention there isprovided a pharmaceutical composition for inducing or accelerating ahealing process of a skin wound, the pharmaceutical compositioncomprising, as an active ingredient, a nucleic acid construct beingdesigned for transforming cells of the skin wound to produce a proteinkinase C, and a pharmaceutically acceptable carrier being designed fortopical application of the pharmaceutical composition.

[0034] According to still a further aspect of the present inventionthere is provided a method of inducing or accelerating a healing processof a skin wound, the method comprising the step of administering to theskin wound a therapeutically effective amount of PKC activator, so as toinduce or accelerate the healing process of the skin wound.

[0035] According to a still further aspect of the present inventionthere is provided a pharmaceutical composition of inducing oraccelerating a healing process of a skin wound, the pharmaceuticalcomposition comprising, as an active ingredient, a therapeuticallyeffective amount of PKC activator, so as to induce or accelerate thehealing process of the skin wound, and an acceptable pharmaceuticalcarrier.

[0036] According to further features in preferred embodiments of theinvention described below, the wound is selected from the groupconsisting of an ulcer, a burn, a laceration and a surgical incision.

[0037] According to still further features in the described preferredembodiments the ulcer is a diabetic ulcer.

[0038] According to still further features in the described preferredembodiments the insulin is recombinant.

[0039] According to still further features in the described preferredembodiments the insulin is of a natural source.

[0040] According to still further features in the described preferredembodiments the additional agent is a platelet-derived growth factor.

[0041] According to still further features in the described preferredembodiments the additional agent is a PKC-α inhibitor.

[0042] According to still further features in the described preferredembodiments administering is effected by a single application.

[0043] According to still further features in the described preferredembodiments the old skin wound is at least 2 days old.

[0044] According to still further features in the described preferredembodiments the insulin has an insulin concentration ranging from 0.1 μMto 10 μM. According to still further features in the described preferredembodiments the dose-unit of insulin is 0.001 to 5 nM in 0.01-0.2 ml ofthe pharmaceutical composition.

[0045] According to still further features in the described preferredembodiments the dose of insulin is ranging from 0.01 to 0.5 nM in0.01-0.2 ml of the pharmaceutical composition.

[0046] According to still further features in the described preferredembodiments the pharmaceutical composition is selected from the groupconsisting of an aqueous solution, a gel, a cream, a paste, a lotion, aspray, a suspension, a powder, a dispersion, a salve and an ointment.

[0047] According to still further features in the described preferredembodiments the pharmaceutical composition includes a solid support.

[0048] According to still further features in the described preferredembodiments the cells are transformed to produce and secrete insulin.

[0049] According to still further features in the described preferredembodiments the cells are transformed by a recombinant PDX1 gene andtherefore the cells produce and secrete natural insulin.

[0050] According to still further features in the described preferredembodiments the cells are transformed by a cis-acting element sequenceintegrated upstream to an endogenous insulin gene of the cells andtherefore the cells produce and secrete natural insulin.

[0051] According to still further features in the described preferredembodiments the insulin secreting cells are capable of forming secretorygranules.

[0052] According to still further features in the described preferredembodiments the insulin secreting cells are endocrine cells.

[0053] According to still further features in the described preferredembodiments the insulin secreting cells are of a human source.

[0054] According to still further features in the described preferredembodiments the insulin secreting cells are of a histocompatibilityhumanized animal source.

[0055] According to still further features in the described preferredembodiments the insulin secreting cells secrete human insulin.

[0056] According to still further features in the described preferredembodiments the insulin secreting cells are autologous cells.

[0057] According to still further features in the described preferredembodiments the cells are selected from the group consisting offibroblasts, epithelial cells and keratinocytes.

[0058] According to still further features in the described preferredembodiments the cells are transformed to produce a protein kinase Ctranscription activator and therefore the cells produce natural proteinkinase C.

[0059] According to still further features in the described preferredembodiments the cells are transformed by a cis-acting element sequenceintegrated upstream to an endogenous protein kinase C of the cells andtherefore the cells produce natural protein kinase C.

[0060] According to still further features in the described preferredembodiments the cells are transformed by a recombinant protein kinase Cgene and therefore the cells produce recombinant protein kinase C.

[0061] According to still further features in the described preferredembodiments the protein kinase C is selected from the group consistingof PKC-β1, PKC-β2, PKC-γ, PKC-θ, PKC-λ, and PKC-ι.

[0062] According to still further features in the described preferredembodiments the protein kinase C is selected from the group consistingof PKC-α, PKC-δ, PKC-ε, PKC-η and PKC-ζ.

[0063] The present invention successfully addresses the shortcomings ofthe presently known configurations by providing new therapeutics tocombat skin wounds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

[0065] In the drawings:

[0066]FIG. 1 demonstrates effective over-expression of PKC isoformsutilizing recombinant adenovirus vectors: Left panel: four day oldprimary keratinocytes were infected for 1 hour utilizing β-galadenovirus 48 hours following infection, cells were fixed and activationof β-galactosidase protein was quantified by the induction of blue colorreaction in comparison to uninfected keratinocytes. Right panel: fourday old primary keratinocytes were infected for 1 hour utilizingrecombinant isoform specific PKC adenoviruses. Twenty four hours later,proteins of infected (Ad) and non infected control (C) cultures wereextracted for Western blot analysis and samples were analyzed usingisoform specific anti-PKC antibodies as described in the Examplessection below.

[0067]FIG. 2 shows that PKC activation by bryostatin 1 inducestranslocation of over-expressed PKC isoforms. Four day old primarykeratinocytes were infected for 1 hour with isoform specific recombinantPKC adenoviruses. Twenty four hours following infection, cells wereeither untreated (C) or stimulated with bryostatin 1 (B) for 30 minutes,and fractionated. Protein samples were subjected to Western blotting andanalyzed using isoform specific anti-PKC antibodies.

[0068]FIG. 3 shows that over-expressed PKC isoforms are active in theirnative form. Four days old primary keratinocytes were infected for 1hour with isoform specific recombinant PKC adenoviruses. Eighteen hoursfollowing infection, cell lysates from uninfected control cells (C) andPKC isoforms over-expressing cells (OE) were immunoprecipitated usingisoform specific anti-PKC antibodies. Immunoprecipitates were subjectedto PKC activity assay as described in the Examples section that follows.

[0069]FIG. 4 demonstrates that over-expression of specific PKC isoformsinduces distinct morphologic changes in primary keratinocytes. Primarykeratinocytes were either left untreated (C) or infected withrecombinant PKC α, δ, η or ζ adenoviruses. Twenty four hours later,cultures were observed by bright field microscopy and photographed(×20).

[0070]FIG. 5 shows distinct localization of over-expressed PKC isoformsin infected primary keratinocytes. Primary keratinocytes were plated onlaminin 5-coated glass slides. Cultures were either untreated orinfected with different recombinant PKC adenoviruses. Twenty four hoursfollowing infection, cells were fixed, washed and air-dried. Cultureswere analyzed by immunofluorescence using isoform specific anti-PKCantibodies, followed by FITC conjugated secondary antibodies. Cells werescanned by confocal microscopy and representative fields werephotographed.

[0071]FIG. 6 demonstrates that PKC isoforms specifically regulate α6β4integrin expression. Five days old primary mouse skin keratinocytes wereuntreated or infected with PKCα, PKCδ, PKCη or PKCζ recombinantadenoviruses. Forty eight hours post infection, membranal cell fractionswere subjected to SDS-PAGE electrophoresis, transferred tonitrocellulose filters, immunoblotted with anti α6 and anti-β4antibodies and analyzed by ECL.

[0072]FIG. 7 shows that over-expression of PKCη and PKCδ induceskeratinocyte proliferation. Five days old primary mouse skinkeratinocytes were untreated or infected with PKCδ, PKCα, PKCη or PKCζrecombinant adenoviruses. Forty eight hours post infection cellproliferation was analyzed by ³H-thymidine incorporation for 1 hour asdescribed in experimental procedures. Results are presented as cpm/dish,in comparison to the β-galactosidase infected keratinocytes. Values arepresented as mean±standard deviation of triplicate determinations in 3separate experiments.

[0073]FIG. 8 demonstrates the PKC isoforms over-expression effects onhemidesmosomal localization of the α6β4 integrin. Primary keratinocyteswere plated on laminin 5 coated glass slides and keratinocyte cultureswere maintained in low Ca²⁺ EMEM for 48 hours. Following that period oftime, cultures were left untreated (A), or infected PKCα, PKCδ, PKCη orPKCζ recombinant adenoviruses (B-E, respectively). Twenty four hourspost infection, keratinocytes were fixed with 4% paraformaldehydefollowed by mild extraction with 0.2% Triton-X-100, washed in PBS andair dried as described in the experimental procedures. Cultures weresubjected to immunofluorescence analysis utilizing isoform specificanti-α6 antibodies, followed by FITC conjugated secondary antibodies, asdescribed in experimental procedures.

[0074] FIGS. 9A-B shows that over-expressed PKCs δ and ζ inducekeratinocyte detachment in vitro. (A)—Primary keratinocytes were eitheruntreated (C) or infected with recombinant PKC α, δ, η or ζadenoviruses. Cell attachment was analyzed 24 and 48 hours followinginfection, by lifting the cells and replating them on matrix coateddishes. Cell counts are presented as protein concentration (mg/dish) ofthe attached cells. (B)—Primary keratinocytes were either untreated (C)or infected with recombinant PKC α, δ, η or ζ adenoviruses. Celldetachment was analyzed 24 hours following infection, by collecting thedetached floating cells in the culture medium. Cell counts are presentedas protein concentration (mg/dish) of the detached cells.

[0075]FIG. 10 demonstrates that PKCη is expressed in activelyproliferating keratinocytes. Primary keratinocytes were plated onlaminin 5-coated glass slides. Forty eight hours following platingkeratinocytes were incubated with BrdU solution for 1 hour followed byimmunofluorescence analysis using anti-PKCη (red) and anti BrdU (green)antibodies as described in the Examples section that follows. Cells werescanned by confocal microscopy and representative fields werephotographed.

[0076]FIG. 11 demonstrates that PKCη induces, while PKCη mutant reduces,keratinocyte proliferation. Primary skin keratinocytes were infected for1 hour with recombinant PKCη or a dominant negative mutant of PKCη(DNPKCη or PKC DNη) adenoviruses. Forty eight hours post infection, cellproliferation was analyzed by 1-hour ³H-thymidine incorporation asdescribed in the Examples section that follows. Results are presented ascpm/dish. Control-uninfected cells.

[0077] FIGS. 12A-B demonstrate that PKCη and DNPKCη over-expressionsspecifically regulate PKC localization and cellular morphology. Primaryskin keratinocytes were infected for 1 hour with recombinant PKCη or adominant negative mutant of PKCη (PKC DNη) adenoviruses. Forty eighthours post infection, keratinocytes were fixed and subjected to (A)bright field photography (×20) and (B) immunofluorescence analysisutilizing PKCη specific antibodies followed by FITC conjugated secondaryantibodies as described in experimental procedures. Control-uninfectedcells.

[0078] FIGS. 13A-B show that inhibition of PKCη expression induceskeratinocyte differentiation in proliferating keratinocytes. Primaryskin keratinocytes were either maintained proliferating in low Ca²⁺medium or differentiated in 0.12 mM Ca²⁺ for 24 hours. Thereafter,keratinocytes were infected for 1 hour with recombinant PKCη or adominant negative mutant of PKCη (PKC DNη) adenoviruses. Twenty fourhours after infection, keratinocytes were either maintained in low Ca²⁺medium or transferred to differentiating medium containing 0.12 mM Ca²⁺for an additional 24 hours. Forty eight hours after infection,keratinocytes were extracted and subjected to SDS-PAGE gels. PKCη (A)and keratin 1 (B) expression was analyzed by Western blotting.

[0079]FIG. 14 demonstrates that topical in vivo expression of PKCηenhances the formation of granulation tissue and accelerates woundhealing in mice incisional wounds. Whole skin 7 mm incisions werecreated on the back of nude mice. Topical application of control β-gal,PKCη and PKCα adenovirus suspension was applied at 1d and 4d followingwounding. Whole skin wounds were fixed in 4% paraformaldehyde and skinsections were analyzed histologically by H&E staining and bright fieldmicroscopy. E—epidermis, D—dermis.

[0080]FIG. 15 demonstrates that insulin, but not IGF1 specificallyinduces translocation of PKCδ in proliferating keratinocytes. Primarykeratinocytes were isolated and plated as described in the Examplessection that follows. Proliferating keratinocytes were maintained forfive days in low Ca²⁺ medium (0.05 mM) until they reached 80%confluency. Cells were stimulated with 10⁻⁷ M insulin (Ins) or 10⁻⁸ MIGF1 (IGF) for 15 minutes. Cells were lysed, as described, and 20 μg ofmembrane or cytosol extracts of stimulated and control unstimulated(Cont) cells were subjected to SDS-PAGE and transfer. Blots were probedwith specific polyclonal antibodies to each PKC isoform.

[0081]FIG. 16 shows that insulin but not IGF1 induces PKCδ activity. Todetermine PKCδ activity, five-day keratinocyte cultures were stimulatedwith 10⁻⁷ M insulin (Ins) or 10⁻⁸ M IGF1 (IGF) for the designated times(1, 15, or 30 minutes). PKCδ was immunoprecipitated from membrane (bluebars, mem) and cytosol (purple bars, cyto) fractions using specificanti-PKCδ antibody. PKCδ immunoprecipitates were analyzed for PKCactivity utilizing an in vitro kinase assay as described in experimentalprocedures. Each bar represents the mean±SE of 3 determinations in 3separate experiments. Values are expressed as pmol ATP/dish/min.

[0082] FIGS. 17A-B show that insulin and IGF1 have an additive effect onkeratinocyte proliferation. Proliferating keratinocytes were maintainedfor five days in low Ca²⁺ medium (0.05 mM) until they reached 80%confluence. (A) Five-day keratinocyte cultures were stimulated for 24hours with insulin or IGF1 at the designated concentrations. (B) Inparallel, keratinocytes were stimulated with 10⁻⁷ M insulin (Ins) andincreasing doses of IGF1 (IGF). At each concentration the right column(striped bar) represents proliferation observed when both hormones wereadded together. The left bar demonstrates the separate effect of 10⁻⁷ Minsulin (red bars) and increasing concentrations of IGF1 (gray bars).Thymidine incorporation was measured as described in experimentalprocedures. The results shown are representative of 6 experiments. Eachbar represents the mean±SE of 3 determinations expressed as percentabove control unstimulated keratinocytes.

[0083] FIGS. 18A-B demonstrate the over-expression of recombinant PKCadenovirus constructs. Keratinocyte cultures were infected utilizingrecombinant adenovirus constructs containing wild type PKCδ (WTPKCδ),wild type PKCα (WTPKCα), or a dominant negative PKCδ mutant (DNPKCδ).(A) Following infection, cells were cultured for 24 hours, harvested,and 20 μg of protein extracts were analyzed by Western blotting usingspecific anti PKCα or anti PKCδ antibodies. The blots presented arerepresentative of 5 separate experiments. (B) Twenty four hoursfollowing infection, cells were harvested and PKCα or PKCδimmunoprecipitates were evaluated by in vitro kinase assay.

[0084]FIG. 19 shows the effects of PKC over-expression on insulin orIGF1-induced proliferation. Non-infected (light blue bars), or cellsover-expressing WTPKCδ (dark blue bars) or DNPKCδ (slashed blue bars)were treated for 24 hours with 10⁻⁷ M insulin (Ins), 10⁻⁸ M IGF1 (IGF)or both (Ins+IGF). Thymidine incorporation was measured as described inexperimental procedures. Each bar represents the mean±SE of 3determinations in 3 experiments done on separate cultures. Values areexpressed as percent of control, unstimulated cells from the sameculture in each experiment.

[0085]FIG. 20 shows that inhibition of PKCδ activity specificallyabrogates insulin induced keratinocyte proliferation. Primarykeratinocytes were cultured as described in the Examples section thatfollows. Non-infected cells or keratinocytes infected with DNPKCδ werestimulated for 24 hours with the following growth factor concentrations:10⁻⁷ M insulin (Ins), 10⁻⁸ M IGF1 (IGF), 10 ng/ml EGF, 10 ng/ml PDGF, 1ng/ml KGF or 5 ng/ml ECGF. Thymidine incorporation was measured asdescribed in the Examples section that follows. Each bar represents themean±SE of 3 determinations in 3 experiments done on separate cultures.Values are expressed as percent of control, unstimulated cells from thesame culture in each experiment.

[0086]FIG. 21 shows that over-expression of PKCδ mediates specificallyinsulin induced keratinocyte proliferation. Primary keratinocytes werecultured as described under FIG. 1. Non-infected cells or keratinocytesinfected with over-expressed WTPKCδ were stimulated for 24 hours withthe following growth factor concentrations: 10⁻⁷ M insulin (Ins), 10⁻⁸ MIGF1 (IGF), 10 ng/ml EGF, 10 ng/ml PDGF, 1 ng/ml KGF or 5 ng/ml ECGF.Thymidine incorporation was measured as described in the Examplessection that follows. Each bar represents the mean±SE of threedeterminations in three experiments done on separate cultures. Valuesare expressed as percent of control, unstimulated cells from the sameculture in each experiment.

[0087] FIGS. 22A-B substantiate the significance of PKCδ and PKCζ in thewound healing process of skin in vivo. Utilizing in vivo mouse model ofnewly developed isofrm specific PKC null mice, PKCα, PKCδ and PKCζ nullmice and their wild type littermates were subjected to a wound healingstudy. Mice were anesthetized and a skin through punch biopsies of 4 mmin diameter were created on the mice back. After a week follow-up, miceskin was removed and skin wound healing was quantified by subjectingskin flaps to a wound strength test utilizing a bursting chambertechnique. Values are expressed as bursting pressure which representsthe maximal pressure within the chamber monitored until bursting occurs.Results represent determinations obtained in distinct groups of 12-20mice. Experiments were repeated at least 3 times.

[0088]FIG. 23 identifies a specific interaction between STAT3 and PKCδin primary skin keratinocytes. Primary keratinocytes were eitheruntreated (upper panel) or infected for 1 hour with isoform specific,recombinant PKC adenoviruses (lower panel). Cells were extracted andimmunoprecipitated (IP) with isoform specific PKC antibodies. Theimmunoprecipitates were subjected to Western blot analysis usinganti-PKCs or anti-STAT3 antibodies.

[0089]FIG. 24 demonstrates the importance of PKCδ activation to insulininduced transcriptional activation of STAT3. Primary keratinocytes wereplated on glass slides and maintained for 5 days in low Ca⁺⁺ medium(0.05 mmol/l) until they reached 80% confluency. Cells were untreated(Cont, upper panel) or pre-treated with 5 μM Rottlerin for 7 minutes (R,lower panel), followed by 10⁻⁷ M insulin for 5 minutes (Ins). Cells werefixed by methanol, washed and air-dried. Cultures were analyzed byimmunofluorescence using antiphospho-Tyr-705-STAT3 antibody, followed byFITC conjugated secondary antibody. Cells were scanned by confocalmicroscopy.

[0090]FIG. 25 demonstrates that overexpression of DN PKCδ inhibitskeratinocyte proliferation induced by overexpression of PKCδ and STAT3.Primary keratinocytes were infected for 1 hour with recombinantadenovirus constructs containing β-Gal (for control), PKC δ, WT STAT3,DN STAT3 or double-infected with DN PKCδ, followed by STAT3. 24 hoursfollowing infection, cell proliferation was analyzed by 1 hour³H-thymidine incorporation. The results are presented as DPM/mg protein.Each bar represents the mean of three determinations in a plate from thesame culture.

[0091]FIG. 26 demonstrates the importance of insulin concentrations andfrequency of applications on wound healing in vivo. Wounds were effectedon the back of 8-10 week old C57BL mice by incision and were treatedwith different concentrations and frequencies of insulin applications(i.e., seven daily repeat applications vs. a single application). Thetreated mice were sacrificed seven days after wounding and the areas oftreated wounds were measured. The results are presented as mm wound areaand each bar represents the mean of six replications±standard deviation(p<0.005).

[0092]FIG. 27 demonstrates histological effects of insulinconcentrations and frequency of applications on wound healing in vivo.Wounds were effected on the back of 8-10 week old C57BL mice by incisionand were treated with different concentrations of insulin andfrequencies of applications (i.e., seven daily repeat applications vs. asingle application). Histological wound sections were performed sevendays after wounding and were analyzed for epidermal and dermal closure(wound contraction). Epidermal closure was assessed by Keratin 14 (K14)antibody staining (left panel) and was considered positive if the woundwas stained positive across the entire gap. The dermal closure wasconsidered positive if both dermal wound sides could be observed under alight microscope in a single field at ×10 magnification (right panel).The results are presented as percent of wound closure over control andeach bar represents the mean of six replications.

[0093]FIG. 28 demonstrates a synergistic effect of combining insulin andplatelet-derived growth factor (PDGF-BB) on wound healing in vivo.Wounds were effected on the back of 8-10 week old C57BL mice by incisionand were treated with a single application of insulin, PDGF-BB, or withinsulin and PDGF-BB combined. The treated mice were sacrificed sevendays after wounding and biopsies were taken for histological analyses ofepidermal and dermal closure (wound contraction). Epidermal closure wasassessed by Keratin 14 (K14) antibody staining (left panel) and wasconsidered positive if the wound was stained positive across the entiregap. The dermal closure was considered positive if both dermal woundsides could be observed under a light microscope in a single field at×10 magnification (right panel). The results are presented as weresummarized in a bar graph as percent of as percent of wound closure overcontrol and each bar represents the mean of six replications.

[0094] FIGS. 29A-D are photographs illustrating the morphological effectof combining insulin and a PKCα inhibitor on wound healing in vivo.Wounds were effected on the back of 8-10 week old C57BL mice by incisionand were treated with insulin (HO/01) combined with a PKCα inhibitor(HO/02). Skin biopsies were removed 7 days after wounding formorphological observations. FIGS. 29A-B show control wounds while FIGS.29C-D show treated wounds.

[0095]FIG. 30 is a histo-micrograph illustrating the combined effect ofinsulin and a PKCα inhibitor on dermal closure (wound contraction).Wounds were effected on the back of 8-10 week old C57BL mice by incisionand were treated daily with insulin (HO/01) combined with a PKCαinhibitor (HO/02). The treated mice were sacrificed seven days afterwounding. Histological wound sections were performed and observed undera light microscope. The dermal closure was considered positive if bothdermal wound sides could be observed in a single ×10 magnification fieldThe opened wound area in the untreated control section (left panel) wastoo large to be contained in a single ×10 magnification field, while thetreated wound section (right panel) shows a positive dermal closure. Theyellow speckled lines mark the dermal edges.

[0096]FIG. 31 is a histo-micrograph illustrating the combined effect ofinsulin and a PKCα inhibitor on epidermal closure. Wounds were effectedon the back of 8-10 week old C57BL mice by incision and were treateddaily with insulin (HO/01) combined with a PKCα inhibitor (HO/02). Thetreated mice were sacrificed seven days after wounding. Histologicalwound sections were performed, stained with keratin 14 (indicative ofepidermal closure) and observed under a light microscope. The openedwound area (arrow marked) in the untreated control section (left panel)was too large to be contained in a single ×10 magnification field, whilethe treated wound section (right panel) shows an epidermal closurethrough the entire wound gap.

[0097]FIG. 32 is a histo-micrograph illustrating the combined effect ofinsulin and a PKCα inhibitor on spatial differentiation of epidermalcells. Wounded mice (C57BL, 8-10 week old) were treated daily withtopical applications of insulin (HO/01) combined with a PKCα inhibitor(HO/02). The treated mice were sacrificed seven days after wounding.Histological wound sections were performed and stained with keratin 1(K1) antibody which highlights the initial stage of spatial celldifferentiation. The untreated control section (left panel) shows a vastundifferentiated wound area (marked by the arrow), while a massiveepidermal reconstruction can be observed in the treated wound section(right panel).

[0098]FIG. 33 demonstrates the quantitative effect of insulin combinedwith a PKCα inhibitor on wound healing in vivo. Wounded mice (C57BL,8-10 week old) were treated daily with topical applications of insulin(HO/01) combined with a PKCα inhibitor (HO/02). The treated mice weresacrificed seven days after wounding. Histological wound sections wereperformed and analyzed for dermal contraction, epidermal closure andspatial differentiation as described in FIGS. 30-32 above. The bar graphshows the incidence (percentage) of fully healed wounds as determined byhistological analyses within each treatment group.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] The present invention is of methods and pharmaceuticalcompositions designed for modulating the expression and/or activation ofserine/threonine protein kinases, also known as PKCs, for inducingand/or accelerating cell proliferation and/or cell differentiation, andthereby accelerate the healing process of wounds. Such modulatedexpression may be effected in accordance with the teachings of thepresent invention by, for example, (i) transformation of wound cellswith a PKC expressing construct; (ii) transformation of wound cells witha cis-acting element to be inserted adjacent to, and upstream of, anendogenous PKC gene of the wound cells; (iii) administration of insulinand other agents acting in synergy with insulin for modulating theexpression and/or activation of PKC in wound cells; (iv) transformationof wound cells with an insulin expressing construct, when expressed andsecreted the insulin produced therefrom serves as an up-regulator forPKC expression and/or activation; (v) transformation of wound cells witha cis-acting element to be inserted adjacent to, and upstream of, theendogenous insulin gene of the wound cells, when expressed and secretedthe insulin serves as an up-regulator for PKC expression and/oractivation; (vi) implantation of insulin secreting cells to the wound;(vii) transformation of wound cells with a trans-acting factor, e.g.,PDX1, for induction of endogenous insulin production and secretion, theinsulin serves as an up-regulator for PKC expression and/or activation;and (viii) administration to the wound of a PKC modulator.

[0100] The principles and operation of the methods and pharmaceuticalcompositions according to the present invention may be better understoodwith reference to the drawings and accompanying descriptions.

[0101] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement of thecomponents set forth in the following description or exemplified in theExamples section. The invention is capable of other embodiments or ofbeing practiced or carried out in various ways. Also, it is to beunderstood that the phraseology and terminology employed herein is forthe purpose of description and should not be regarded as limiting.

[0102] Adult skin includes two layers: a keratinized stratifiedepidermis and an underlying thick layer of collagen-rich dermalconnective tissue providing support and nourishment. Skin serves as theprotective barrier against the outside world. Therefore any injury orbreak in the skin must be rapidly and efficiently mended. As describedin the Background section hereinabove, the first stage of the repair isachieved by formation of the clot that plugs the initial wound.Thereafter, inflammatory cells, fibroblasts and capillaries invade theclot to form the granulation tissue. The following stages involvere-epithelization of the wound where basal keratinocytes have to losetheir hemidesmosomal contacts, keratinocytes migrate upon thegranulation tissue to cover the wound. Following keratinocyte migration,keratinocytes enter a proliferative boost, which allows replacement ofcells lost during the injury. After the wound is covered by a monolayerof keratinocytes, new stratified epidermis is formed and the newbasement membrane is reestablished (20-23). Several growth factors havebeen shown to participate in this process including EGF family of growthfactors, KGF, PDGF and TGFβ1 (22-24). Among these growth factors bothEGF and KGF are thought to be intimately involved in the regulation ofproliferation and migration of epidermal keratinocytes (25,26).Fundamental to the understanding of wound healing biology is a knowledgeof the signals that trigger the cells in the wound to migrate,proliferate, and lay down new matrix in the wound gap.

[0103] To facilitate understanding of the invention set forth in thedisclosure that follows, a number of terms are defined below.

[0104] The term “wound” refers broadly to injuries to the skin andsubcutaneous tissue initiated in any one of a variety of ways (e.g.,pressure sores from extended bed rest, wounds induced by trauma, cuts,ulcers, burns and the like) and with varying characteristics. Wounds aretypically classified into one of four grades depending on the depth ofthe wound: (i) Grade I: wounds limited to the epithelium; (ii) Grade II:wounds extending into the dermis; (iii) Grade III: wounds extending intothe subcutaneous tissue; and (iv) Grade IV (or full-thickness wounds):wounds wherein bones are exposed (e.g., a bony pressure point such asthe greater trochanter or the sacrum).

[0105] The term “partial thickness wound” refers to wounds thatencompass Grades I-III; examples of partial thickness wounds includeburn wounds, pressure sores, venous stasis ulcers, and diabetic ulcers.

[0106] The term “deep wound” is meant to include both Grade III andGrade IV wounds.

[0107] The term “healing” in respect to a wound refers to a process torepair a wound as by scar formation.

[0108] The phrase “inducing or accelerating a healing process of a skinwound” refers to either the induction of the formation of granulationtissue of wound contraction and/or the induction of epithelialization(i.e., the generation of new cells in the epithelium). Wound healing isconveniently measured by decreasing wound area.

[0109] The present invention contemplates treating all wound types,including deep wounds and chronic wounds.

[0110] The term “chronic wound” refers to a wound that has not healedwithin thirty days.

[0111] The phrase “transforming cells” refers to a transient orpermanent alteration of a cell's nucleic acid content by theincorporation of exogenous nucleic acid which either integrates into thecell genome and genetically modifies the cell or remains unintegrated.

[0112] The term “cis-acting element” is used herein to describe agenetic region that serves as an attachment site for DNA-bindingproteins (e.g., enhancers, operators and promoters) thereby affectingthe activity of one or more genes on the same chromosome.

[0113] The phrase “trans-acting factor” is used herein to describe afactor that binds to a cis-acting element and modulates its activitywith respect to gene expression therefrom. Thus, PDX1 is a trans-actingfactor which binds to the insulin gene promoter and modulates itsactivity.

[0114] The phrase “transcription activator” is used herein to describe afactor that increases gene expression. A trans-acting factor is anexample of a direct transcription activator.

[0115] The term “activator” is used herein to describe a molecule thatenhances an activity.

[0116] The phrase “modulated expression and/or activation” used hereinrefers to enhanced or inhibited expression and/or activation.

[0117] PKC is a major signaling pathway, which mediates keratinocyteproliferation and differentiation. PKC isoforms α, δ, ε, η and ζ areexpressed in the skin (4, 10). While conceiving the present invention itwas hypothesized that PKC modulated expression and/or activation mayinduce cell proliferation and/or cell differentiation and therebyaccelerate the healing process of wounds. While reducing the presentinvention to practice this theory has been approved by numerousexperiments showing that PKC modulated expression and/or activationindeed induces cell proliferation and cell differentiation andaccelerates the healing process of wounds. As is further delineatedherein in great detail, various distinct approaches were undertaken tomodulates expression and/or activation of PKC to thereby accelerate thehealing process of wounds. Based on the experimental findings, otherapproaches have been devised. A striking and novel phenomenon wasdiscovered while reducing the present invention to practice-insulinserves as a modulator of expression and/or activation of PKC. As such,insulin may serve as a therapeutic agent for modulating the expressionand/or activation of PKC so as to accelerate the healing process ofwounds.

[0118] The characteristics of distinct PKC isoforms and their specificeffects on cell proliferation and/or differentiation are of greatimportance to the biology of skin wound healing. Utilizing PKCadenovirus constructs enabled to identify the specific roles of avariety of PKC isoforms in the wound healing process in vitro and invivo. All isoforms were able to specifically affect different aspects ofkeratinocyte growth and differentiation. Two isoforms, PKCδ and PKCζ,could specifically regulate integrin regulation (see Example 6 below),adherence to the basement membrane (see Example 9 below) andhemidesmosome formation (see Example 8 below). Two isoforms, PKCδ andPKCη, were found to regulate the proliferation potential of epidermalkeratinocytes (see Examples 7 and 11 below). In addition, a dominantnegative isoform of PKCη (DNPKCη) was able to specifically inducedifferentiation in actively proliferating keratinocytes (see Example 12below). Finally, the importance of distinct PKC isoforms to the woundhealing process in skin was also verified in an in vivo system.Utilizing PKC null mice where expression of distinct PKC isoforms wasabolished it is shown herein that PKCδ and PKCζ which were found to berequired for both adhesion and motility processes in skin keratinocytesare also important in the in vivo wound healing process in an animalmodel (see Example 19). Whole skin full thickness biopsies in PKC nullskin suggested that both PKCδ and PKCζ but not PKCα are essential forproper healing of the wound. Furthermore, Example 22 below shows that aPKCα inhibitor effectively promoted wound healing in vivo thusindicating that the PKCα isoform may be antagonistic to wound healing.

[0119] PKCη has a unique tissue distribution. It is predominantlyexpressed in epithelial tissues (27,28). In situ hybridization studiesas well as immunohistochemical studies have demonstrated PKCη is highlyexpressed in the differentiating and differentiative layers (27). Theresults presented herein suggest the role of PKCη as a functionalregulator of both proliferation and differentiation of skin depending onthe cellular physiology. When keratinocytes are maintained in aproliferative state under low Ca²⁺ conditions, PKCη induced theproliferation rate five to seven times above control keratinocytes.However, when cells were induced to differentiate by elevating the Ca²⁺concentration, differentiation was induced in a faster and higher ratein comparison to control cells (see Example 12). This could explain theability of PKCη to dramatically induce wound healing and formation ofgranulation tissue as both proliferative capacity and formation ofdifferentiation layers were achieved. Interestingly, the wound healingresults in vivo and the expression of PKCη in embryonic tissue, whichnormally does not express PKCη at high levels in adulthood, wouldsuggest a possible role for PKCη in the proliferation and tissueorganization of other tissues as well. This includes neuronal as well asdermal and muscle tissue, which were efficiently healed in thegranulation tissue of the wound. Furthermore, the ability tospecifically regulate differentiation of keratinocytes and induce normaldifferentiation in actively proliferating cells by utilizing a dominantnegative mutant allows specifically to manipulate differentiation andcontrol hyperproliferative disorders involved in wound healing.

[0120] It is exemplified herein that the healing ability of PKCη isexerted in vivo, on wounds that were produced on the backs of nude mice.Example 14 below shows that administration of PKCη expressing constructto the wound resulted in a granulation tissue formation, four days aftertopical infection.

[0121] Overall, the results presented herein demonstrate that modulatingexpression and/or activation (membrane mobilization) of distinct PKCisoforms is an effective tool to combat wounds. Accordingly, woundhealing may be promoted by enhancing the expression and/or activity ofisoforms PKCδ, PKCη and PKCζ, or by inhibiting the expression and/oractivity of isoform PKCα.

[0122] Thus, according to one aspect of the present invention there isprovided a method of inducing or accelerating a healing process of askin wound, the method is effected by administering to the skin wound atherapeutically effective amount of at least one agent for modulatingPKC production and/or activation. A pharmaceutical composition foreffecting the method according to this aspect of the present inventiontherefore includes, as an active ingredient, a therapeutically effectiveamount of at least one agent for modulating PKC production and/oractivation; and a pharmaceutically acceptable carrier.

[0123] Skin is not considered to be a classic insulin responsive tissue.Therefore, the effects of insulin in skin are mostly attributed to itsability to activate the closely related IGFR. It was shown that inkeratinocytes, both insulin and IGF1 can stimulate both receptors andactivate similar downstream effectors (34). However, the presentinvention demonstrates that whereas both growth factors inducekeratinocyte proliferation in a dose-dependent manner, each hormoneexerts its effects through distinct signaling pathways. The initialindication for differential regulation of keratinocyte proliferation byinsulin and IGF1 was confirmed by the finding that these hormones had anadditive effect on keratinocyte proliferation when added together, atmaximal proliferation-inducing concentration of each hormone (seeExample 15). In order to identify the divergence point in insulin andIGF1 signaling pathway in regulation of keratinocyte proliferation,elements known to both regulate keratinocyte proliferation and to act asdownstream effectors of insulin signaling were examined. These studiesrevealed that insulin signaling is specifically mediated by PKCδ inkeratinocyte proliferation (see Example 17). PKCδ is a unique isoformamong the PKC family of proteins involved specifically in growth andmaturation of various cell types (35). However, while PKCδ was shown tobe specifically regulated by stimulation of several growth factorsincluding EGF, Platelet derived growth factor and neurotransmitters, itsphysiological effects were shown to participate in growth factorinhibition of cell growth including apoptosis, differentiation, and cellcycle retardation or arrest (36-41). Recently it was shown that within12-24 hours after elevation of Ca²⁺, a selective loss of the α6β4integrin complex is linked to induction of the K1 in cultured mousekeratinocytes (6). The loss of α6β4 protein expression is a consequenceof transcriptional and post-translational events including enhancedprocessing of the α6 and β4 chains. In preliminary studies a link wasestablished between the activation of PKC and the processing andregulation of the α6β4 integrin. These results are in agreement withprevious results on the role of PKCδ as well as PKCζ in loss of α6β4expression and hemidesmosome formation inducing keratinocyte detachment.However, the present invention identifies another role for PKCδ, as atarget for insulin induced keratinocyte proliferation. The examplesbelow show that only insulin stimulation, but not a variety of growthfactors, including, but not limited to, EGF, KGF, PDGF, ECGF and IGF1,can translocate and activate PKCδ, but not any of the other PKC isoformsexpressed in skin. The importance of PKCδ to insulin stimulation wasfurther confirmed when the mitogenic stimulation by EGF, KGF, PDGF, ECGFand IGF1 were not abrogated by the dominant negative mutant of PKCδ andinsulin appeared to be the primary activator of this PKC isoform in theregulation of keratinocyte proliferation (see Example 17). However, whenkeratinocytes were infected with WT PKCδ keratinocytes mitogenicstimulation by EGF and KGF was enhanced. This suggests that PKCδactivation is also essential for the proliferative stimulation of othergrowth factors by upstream signaling pathways. Moreover, down streamelements were characterized which meidate in insulin induced PKCδactivation and keratinocyte proliferation and the involvement of STAT3,a transcriptional activator in this process, was identified. STAT(Signal Transducers and Activators of Transcription) proteins are afamily of transcription factors recruited by a variety of cytokines andgrowth factors. Among the seven known STAT family members STAT3 isunique. Targeted disruption of STAT3 but not other STAT family membersleads to early embryonic lethality. Specifically, when STAT3 wasconditionally ablated in skin, skin remodeling was severely disrupted.Upon activation, STAT proteins form homo or heterodimers, translocate tothe nucleus and bind to DNA response elements of target genes to inducetranscription. It was found that in keratinocytes, PKCδ but not otherPKC isoforms expressed in skin (PKCs α, ζ, η and ε) is constitutivelyassociated with STAT3 (see, Example 18). Furthermore, insulin regulatesphosphorylation, activation and nuclear translocation of STAT3 viaspecific activation of PKCδ. Inhibition of PKCδ activity by apharmacological inhibitor, rottlerin or by overexpressing a dominantnegative PKCδ mutant abrogated insulin induced STAT3 activation andnuclear translocation. Finally, overexpression of a dominant negativePKCδ mutant inhibited keratinocyte proliferation induced byoverexpression of STAT3 (see, Example 18). These results suggest a rolefor insulin induced PKCδ activity in transcriptional activation by STAT3in skin keratinocyte proliferation. As STAT3 is important for skinremodeling and is a down stream effector recruited by a variety ofcytokines and growth factors, overall these results suggest PKCδactivation as a primary downstream element mediating the proliferationof keratinocytes by a variety of skin growth factors. Specifically, PKCδcould be the primary candidate for the pathogenesis of defective woundhealing as it appears in diabetic patients. The link between PKCδ andwound healing was also been coroborated in vivo. Utilizing a newlyconstructed PKCδ null mouse it is shown herein that the lack of PKCδ,delays wound healing in mice skin (see Example 19). The link betweenPKCδ and insulin signaling has also been established in several othersystems. For example, it was recently shown that in muscle cultures,PKCδ mediates insulin-induced glucose transport (42, 43). Similarly, incells over-expressing the insulin receptor, insulin stimulation wasshown to be associated with activation of PKCδ (44-46). However, whereasin these studies insulin mediated PKCδ activation has been linked to themetabolic effects of insulin, this is the first report linking PKCδ toinsulin mediated cell proliferation. An identified dual role for PKCδ inregulation of both keratinocytes proliferation and the control of theearly differentiation stages where cells lose their adherence to theunderlying basement membrane was shown. This would suggest insulininduced PKCδ as a primary candidate of regulation of physiologicalbalance between proliferation and differentiation in skin.

[0124] Thus, in accordance with the teachings of the present inventionmodulating PKC production and/or activation is effected by subjectingwound cells to insulin. This can be executed by one of a plurality ofalternative ways as is further exemplified hereinunder.

[0125] One way is the direct administration of insulin to the wound. Asdescribed in Examples 21 and 22 hereinbelow, a topical application ofinsulin on wounds at a concentration ranging from 0.1-10 μM effectivelypromoted epidermal and dermal closure and subsequently wound healing.Yet, surprisingly and unexpectedly, the application of insulin combinedwith PDGF-BB growth factor, or with a PKCα inhibitor, resulted in asubstantial and synergetic improvement of the wound healing process overthe insulin alone. Thus, according to another aspect of the presentinvention there is provided a method of inducing or accelerating ahealing process of a skin wound. The method is effected by administeringto the skin wound a therapeutically effective amount of insulin and atleast one additional agent acting in synergy with the insulin, so as toinduce or accelerate the healing process of the skin wound. Preferably,the agent is a PKCα inhibitor. Further preferably, the agent is a growthfactor such as PDGF, EGF, TGFβ, KGF, ECGF or IGF1, and most preferablythe agent is PDGF-BB.

[0126] The direct administration of insulin, either alone or combinedwith another agent, may be effected by a single or by repeatapplications. While reducing the present invention to practice, theinventors surprisingly discovered that a treatment with a singleapplication of insulin at a concentration of 1 μM was substantially moreeffective in healing wounds than with seven repeat daily applications ofinsulin at a similar concentration (see Example 20 below). Thus,according to another aspect of the present invention, there is provideda method of inducing or accelerating a healing process of a skin woundby administering to the skin wound a single dose-unit of atherapeutically effective amount of insulin. Preferably the singledose-unit comprises 0.001 to 5 nM, preferably 0.01 to 0.5 nM of insulinin, for example, an aqueous solution, gel, cream, paste, lotion, spray,suspension, powder, dispersion, salve or ointment formulation in anamount sufficient to cover a 1 cm area of the skin wound, e.g., 0.01-0.2ml.

[0127] The timing of administering insulin onto wounds may be critical,as illustrated in Example 20 in the Examples section that follows. Forexample, a single application of insulin to a 4 days-old wound resultedin effective wound healing. Thus, according to another aspect of thepresent invention, there is provided a method of inducing oraccelerating a healing process of an old skin wound by administering tothe wound a single dose of a therapeutically effective amount ofinsulin.

[0128] The phrase “old skin wound” used herein refers to a skin woundthat is at least one day old, at least two days old, at least three daysold, preferably, at least four days old.

[0129] A pharmaceutical composition for inducing or accelerating ahealing process of a skin wound, according to another aspect of thepresent invention, includes, as an active ingredient, a therapeuticallyeffective amount of insulin, at least one additional agent acting insynergy with the insulin, and a pharmaceutically acceptable carrierdesigned for topical application of the pharmaceutical composition.Preferably, the agent is a PKCα inhibitor or a growth factor such asPDGF, EGF, TGFβ, KGF, ECGF or IGF1, and most preferably PDGF-BB. Thepharmaceutically acceptable carrier can be, but not limited to, a gel, acream, a paste, a lotion, a spray, a suspension, a powder, a dispersion,a salve and an ointment, as is further detailed hereinunder. Solidsupports can also be used for prolonged release of insulin into thewound. It will be appreciated that the insulin can be native orpreferably recombinant, of a human or any other suitable source.

[0130] According to another aspect of the present invention, apharmaceutical composition for inducing or accelerating a healingprocess of a skin wound, may include a single dose-unit of insulinselected capable of inducing or accelerating a healing process of theskin wound, and a pharmaceutically acceptable carrier being designed fortopical application of the pharmaceutical composition. Preferably, thesingle dose-unit of insulin is ranging from 0.001 to 5 nM, preferably0.01 to 0.5 nM, in a 0.01-0.2 ml formulation dose-unit.

[0131] In an alternative embodiment of the present invention, cellsexpressing and secreting insulin are implanted into the wound, so as toinduce or accelerate the healing process of the skin wound. Such insulinproducing cells may be cells naturally producing insulin, oralternatively, such cells are transformed to produce and secreteinsulin. The cells can be transformed by, for example, a recombinantPDX1 gene (see GeneBank Accession Nos. AH005712, AF035260, AF035259)which is a trans-acting factor for the production and secretion ofinsulin. Alternatively, the cells can be transformed by a cis-actingelement sequence, such as a strong and constitutive or induciblepromoter integrated upstream to an endogenous insulin gene of the cells,by way of gene knock-in, so as to transform the cells to overproduce andsecrete natural insulin. This is obtainable because the upstream regionsof the insulin gene have been cloned (See Accession Nos. E00011,NM000207). Alternatively, the cells are transformed by a recombinantinsulin gene (e.g., Accession No. J02547) and therefore the cellsproduce and secrete recombinant insulin.

[0132] A pharmaceutical composition for inducing or accelerating ahealing process of a skin wound according to this aspect of the presentinvention therefore includes, as an active ingredient, insulin secretingcells, and a pharmaceutically acceptable carrier which is designed fortopical application of the pharmaceutical composition. Advantageously,the insulin secreting cells administered to a wound are capable offorming secretory granules, so as to secrete insulin produced thereby.The insulin secreting cells can be endocrine cells. They can be of ahuman source or of a histocompatibility humanized animal source. Mostpreferably, the insulin secreting cells, either transformed or not, areof an autologous source. The insulin secreted by the insulin secretingcells is preferably human insulin or has the amino acid sequence ofhuman insulin. The insulin secreting cells can be fibroblasts,epithelial cells or keratinocytes, provided that a transformation asdescribed above is employed so as to render such cells to produce andsecrete insulin.

[0133] In still an alternative embodiment, cells of the skin wound aretransformed to produce and secrete insulin, so as to induce oraccelerate the healing process of the skin wound.

[0134] A pharmaceutical composition for inducing or accelerating ahealing process of a skin wound according to this aspect of the presentinvention therefore includes, as an active ingredient, a nucleic acidconstruct designed for transforming cells of the skin wound to produceand secrete insulin, and a pharmaceutically acceptable carrier designedfor topical application of the pharmaceutical composition.

[0135] Any one of the transformation methods described above, e.g.,transformation with a construct encoding insulin, transformation with aconstruct harboring a cis-acting element for insertion downstream of anendogenous insulin gene by way of gene knock-in, and transformation witha construct encoding a trans-acting factor for activation of endogenousinsulin production and secretion, can be employed in context of thisembodiment of the present invention.

[0136] Previous studies on the effects of distinct PKC isoforms in skinhave been hampered as a result of the difficulty in introducing foreigngenes efficiently into primary cells by conventional methods due to theshort life span, differentiation potential and the inability to isolatestable transformants. To overcome these obstacles, viral vectors arebeing used to introduce genes of interest. Viral vectors are developedby modification of the viral genome in the form of replicative defectiveviruses. The most widely used viral vectors are the retroviruses andadenoviruses, which are used for experimental as well as gene therapypurposes (13). Specifically, the high efficiency of adenovirus infectionin non replicating cells, the high titer of virus and the highexpression of the transduced protein makes this system highlyadvantageous to primary cultures compared to retroviral vectors. Asadenoviruses do not integrate into the host genome and the stable viraltiters can be rendered replication deficient, these viral constructs areassociated with minimal risk for malignancies in human as well as animalmodels (14). To date, in skin, adenovirus constructs have also been usedsuccessfully with high efficiency of infection with ex vivo and in vivoapproaches (15, 16). An adenovirus vector, which was developed by I.Saito and his associates (17) was used in the present study. The cosmidcassette (pAxCAwt) has nearly a full length adenovirus 5 genome butlacks E1A, E1B and E3 regions, rendering the virus replicationdefective. It contains a composite CAG promoter, consisting of thecytomegalovirus immediate-early enhancer, chicken β-actin promoter, anda rabbit β-globin polyadenylation signal, which strongly inducesexpression of inserted DNAs (13, 17). A gene of interest is insertedinto the cosmid cassette, which is then co-transfected into humanembryonic kidney 293 cells together with adenovirus DNA terminal proteincomplex (TPC). In 293 cells that express E1A and E1B regions,recombination occurs between the cosmid cassette and adenovirus DNA-TPC,yielding the desired recombinant virus at an efficiency one hundred foldthat of conventional methods. Such high efficiency is mainly due to theuse of the adenovirus DNA-TPC instead of proteinesed DNA. Furthermore,the presence of longer homologous regions increases the efficiency ofthe homologous recombination. Regeneration of replication competentviruses is avoided due to the presence of multiple EcoT221 sites. Itshould be noted in this respect that keratinocytes were infected withdistinct PKC recombinant adenoviruses, demonstrated 24 hours latereffective over-expression of PKC isoforms (see example 1).

[0137] Thus, another way by which modulating PKC production and/oractivation is effected according to the present invention is by inducingover-expression of a PKC in the skin wound cells. This can be achievedby transforming the cells with a cis-acting element sequence integrated,by way of homologous recombination, upstream to an endogenous proteinkinase C of the cells and thereby causing the cells to produce naturalprotein kinase C. Still alternatively, this can be achieved bytransforming the cells with a recombinant protein kinase C gene, suchas, but not limited to, PKC-β1 gene (Accession Nos. X06318, NM002738),PKC-β2 gene (Accession No. X07109), PKC-γ gene (Accession No. L28035),PKC-θ gene (Accession No. L07032), PKC-λ gene (Accession No. D28577),PKC-ι gene (Accession No. L18964), PKC-α gene (Accession No. X52479),PKC-δ gene (Accession Nos. L07860, L07861), PKC-ε gene (Accession No.X72974), PKC-η gene (Accession No. Z15108) and PKC-ζ gene (AccessionNos. Z15108, X72973, NM002744), and thereby causing the cells to producerecombinant protein kinase C.

[0138] A pharmaceutical composition for inducing or accelerating ahealing process of a skin wound according to this aspect of the presentinvention therefore includes, as an active ingredient, a nucleic acidconstruct designed for transforming cells of the skin wound to produce aprotein kinase C, and a pharmaceutically acceptable carrier designed fortopical application of the pharmaceutical composition.

[0139] Still another way by which modulating PKC production and/oractivation is effected according to the present invention is by a PKCactivator, such as, but not limited to Ca²⁺, insulin or bryostatin 1, soas to induce or accelerate the healing process of the skin wound.

[0140] A pharmaceutical composition of inducing or accelerating ahealing process of a skin wound according to this aspect of the presentinvention therefore includes, as an active ingredient, a therapeuticallyeffective amount of a PKC activator, so as to induce or accelerate thehealing process of the skin wound, and an acceptable pharmaceuticalcarrier.

[0141] The therapeutically/pharmaceutically active ingredients of thepresent invention can be administered to a wound per se, or in apharmaceutical composition mixed with suitable carriers and/orexcipients. Pharmaceutical compositions suitable for use in context ofthe present invention include those compositions in which the activeingredients are contained in an amount effective to achieve an intendedtherapeutic effect.

[0142] As used herein a “pharmaceutical composition” refers to apreparation of one or more of the active ingredients described herein,either protein, chemicals, nucleic acids or cells, or physiologicallyacceptable salts or prodrugs thereof, with other chemical componentssuch as traditional drugs, physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound or cell to an organism. Pharmaceuticalcompositions of the present invention may be manufactured by processeswell known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

[0143] Hereinafter, the phrases “physiologically suitable carrier” and“pharmaceutically acceptable carrier” are interchangeably used and referto a carrier or a diluent that does not cause significant irritation toan organism and does not abrogate the biological activity and propertiesof the administered conjugate.

[0144] Herein the term “excipient” refers to an inert substance added toa pharmaceutical composition to further facilitate processes andadministration of the active ingredients. Examples, without limitation,of excipients include calcium carbonate, calcium phosphate, varioussugars and types of starch, cellulose derivatives, gelatin, vegetableoils and polyethylene glycols.

[0145] Techniques for formulation and administration of activeingredients may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., latest edition, which is incorporatedherein by reference.

[0146] While various routes for the administration of active ingredientsare possible, and were previously described, for the purpose of thepresent invention, the topical route is preferred, and is assisted by atopical carrier. The topical carrier is one, which is generally suitedfor topical active ingredients administration and includes any suchmaterials known in the art. The topical carrier is selected so as toprovide the composition in the desired form, e.g., as a liquid ornon-liquid carrier, lotion, cream, paste, gel, powder, ointment,solvent, liquid diluent, drops and the like, and may be comprised of amaterial of either naturally occurring or synthetic origin. It isessential, clearly, that the selected carrier does not adversely affectthe active agent or other components of the topical formulation, andwhich is stable with respect to all components of the topicalformulation. Examples of suitable topical carriers for use hereininclude water, alcohols and other nontoxic organic solvents, glycerin,mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetableoils, parabens, waxes, and the like. Preferred formulations herein arecolorless, odorless ointments, liquids, lotions, creams and gels.

[0147] Ointments are semisolid preparations, which are typically basedon petrolatum or other petroleum derivatives. The specific ointment baseto be used, as will be appreciated by those skilled in the art, is onethat will provide for optimum active ingredients delivery, and,preferably, will provide for other desired characteristics as well,e.g., emolliency or the like. As with other carriers or vehicles, anointment base should be inert, stable, nonirritating and nonsensitizing.As explained in Remington: The Science and Practice of Pharmacy, 19thEd. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399-1404,ointment bases may be grouped in four classes: oleaginous bases;emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginousointment bases include, for example, vegetable oils, fats obtained fromanimals, and semisolid hydrocarbons obtained from petroleum.Emulsifiable ointment bases, also known as absorbent ointment bases,contain little or no water and include, for example, hydroxystearinsulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointmentbases are either water-in-oil (W/O) emulsions or oil-in-water (O/W)emulsions, and include, for example, cetyl alcohol, glycerylmonostearate, lanolin and stearic acid. Preferred water-soluble ointmentbases are prepared from polyethylene glycols of varying molecularweight; again, reference may be made to Remington: The Science andPractice of Pharmacy for further information.

[0148] Lotions are preparations to be applied to the skin surfacewithout friction, and are typically liquid or semiliquid preparations,in which solid particles, including the active agent, are present in awater or alcohol base. Lotions are usually suspensions of solids, andmay comprise a liquid oily emulsion of the oil-in-water type. Lotionsare preferred formulations herein for treating large body areas, becauseof the ease of applying a more fluid composition. It is generallynecessary that the insoluble matter in a lotion be finely divided.Lotions will typically contain suspending agents to produce betterdispersions as well as compounds useful for localizing and holding theactive agent in contact with the skin, e.g., methylcellulose, sodiumcarboxymethylcellulose, or the like.

[0149] Creams containing the selected active ingredients are, as knownin the art, viscous liquid or semisolid emulsions, either oil-in-wateror water-in-oil. Cream bases are water-washable, and contain an oilphase, an emulsifier and an aqueous phase. The oil phase, also sometimescalled the “internal” phase, is generally comprised of petrolatum and afatty alcohol such as cetyl or stearyl alcohol; the aqueous phaseusually, although not necessarily, exceeds the oil phase in volume, andgenerally contains a humectant. The emulsifier in a cream formulation,as explained in Remington, supra, is generally a nonionic, anionic,cationic or amphoteric surfactant.

[0150] Gel formulations are preferred for application to the scalp. Aswill be appreciated by those working in the field of topical activeingredients formulation, gels are semisolid, suspension-type systems.Single-phase gels contain organic macromolecules distributedsubstantially uniformly throughout the carrier liquid, which istypically aqueous, but also, preferably, contains an alcohol and,optionally, an oil.

[0151] Carriers for nucleic acids include, but are not limited to,liposomes including targeted liposomes, nucleic acid complexing agents,viral coats and the like. However, transformation with naked nucleicacids may also be used.

[0152] Various additives, known to those skilled in the art, may beincluded in the topical formulations of the invention. For example,solvents may be used to solubilize certain active ingredientssubstances. Other optional additives include skin permeation enhancers,opacifiers, anti-oxidants, gelling agents, thickening agents,stabilizers, and the like.

[0153] As has already been mentioned hereinabove, topical preparationsfor the treatment of wounds according to the present invention maycontain other pharmaceutically active agents or ingredients, thosetraditionally used for the treatment of such wounds. These includeimmunosuppressants, such as cyclosporine, antimetabolites, such asmethotrexate, corticosteroids, vitamin D and vitamin D analogs, vitaminA or its analogs, such etretinate, tar, coal tar, anti pruritic andkeratoplastic agents, such as cade oil, keratolytic agents, such assalicylic acid, emollients, lubricants, antiseptics and disinfectants,such as the germicide dithranol (also known as anthralin)photosensitizers, such as psoralen and methoxsalen and UV irradiation.Other agents may also be added, such as antimicrobial agents, antifungalagents, antibiotics and anti-inflammatory agents. Treatment byoxygenation (high oxygen pressure) may also be co-employed.

[0154] The topical compositions of the present invention may also bedelivered to the skin using conventional dermal-type patches orarticles, wherein the active ingredients composition is contained withina laminated structure, that serves as a drug delivery device to beaffixed to the skin. In such a structure, the active ingredientscomposition is contained in a layer, or “reservoir”, underlying an upperbacking layer. The laminated structure may contain a single reservoir,or it may contain multiple reservoirs. In one embodiment, the reservoircomprises a polymeric matrix of a pharmaceutically acceptable contactadhesive material that serves to affix the system to the skin duringactive ingredients delivery. Examples of suitable skin contact adhesivematerials include, but are not limited to, polyethylenes, polysiloxanes,polyisobutylenes, polyacrylates, polyurethanes, and the like. Theparticular polymeric adhesive selected will depend on the particularactive ingredients, vehicle, etc., i.e., the adhesive must be compatiblewith all components of the active ingredients-containing composition.Alternatively, the active ingredients-containing reservoir and skincontact adhesive are present as separate and distinct layers, with theadhesive underlying the reservoir which, in this case, may be either apolymeric matrix as described above, or it may be a liquid or hydrogelreservoir, or may take some other form.

[0155] The backing layer in these laminates, which serves as the uppersurface of the device, functions as the primary structural element ofthe laminated structure and provides the device with much of itsflexibility. The material selected for the backing material should beselected so that it is substantially impermeable to the activeingredients and to any other components of the activeingredients-containing composition, thus preventing loss of anycomponents through the upper surface of the device. The backing layermay be either occlusive or nonocclusive, depending on whether it isdesired that the skin become hydrated during active ingredientsdelivery. The backing is preferably made of a sheet or film of apreferably flexible elastomeric material. Examples of polymers that aresuitable for the backing layer include polyethylene, polypropylene, andpolyesters.

[0156] During storage and prior to use, the laminated structure includesa release liner. Immediately prior to use, this layer is removed fromthe device to expose the basal surface thereof, either the activeingredients reservoir or a separate contact adhesive layer, so that thesystem may be affixed to the skin. The release liner should be made froman active ingredients/vehicle impermeable material.

[0157] Such devices may be fabricated using conventional techniques,known in the art, for example by casting a fluid admixture of adhesive,active ingredients and vehicle onto the backing layer, followed bylamination of the release liner. Similarly, the adhesive mixture may becast onto the release liner, followed by lamination of the backinglayer. Alternatively, the active ingredients reservoir may be preparedin the absence of active ingredients or excipient, and then loaded by“soaking” in an active ingredients/vehicle mixture.

[0158] As with the topical formulations of the invention, the activeingredients composition contained within the active ingredientsreservoirs of these laminated systems may contain a number ofcomponents. In some cases, the active ingredients may be delivered“neat,” i.e., in the absence of additional liquid. In most cases,however, the active ingredients will be dissolved, dispersed orsuspended in a suitable pharmaceutically acceptable vehicle, typically asolvent or gel. Other components, which may be present, includepreservatives, stabilizers, surfactants, and the like.

[0159] The pharmaceutical compositions herein described may alsocomprise suitable solid or gel phase carriers or excipients. Examples ofsuch carriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

[0160] Dosing is dependent on the type, the severity and manifestationof the affliction and on the responsiveness of the subject to the activeingredients, as well as the dosage form employed, the potency of theparticular conjugate and the route of administration utilized. Personsof ordinary skill in the art can easily determine optimum dosages,dosing methodologies and repetition rates. The exact formulation, routeof administration and dosage can be chosen by the individual physicianin view of the patient's condition. (See e.g., Fingl, et al., 1975, in“The Pharmacological Basis of Therapeutics”, Ch. 1 p.1).

[0161] Thus, depending on the severity and responsiveness of thecondition to be treated, dosing can be a single or repetitiveadministration, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the skin lesionis achieved.

[0162] In some aspects the present invention utilizes in vivo and exvivo (cellular) gene therapy techniques which involve celltransformation and gene knock-in type transformation. Gene therapy asused herein refers to the transfer of genetic material (e.g. DNA or RNA)of interest into a host to treat or prevent a genetic or acquireddisease or condition or phenotype. The genetic material of interestencodes a product (e.g., a protein, polypeptide, peptide, functionalRNA, antisense RNA) whose production in vivo is desired. For example,the genetic material of interest can encode a hormone, receptor, enzyme,polypeptide or peptide of therapeutic value. For review see, in general,the text “Gene Therapy” (Advanced in Pharmacology 40, Academic Press,1997).

[0163] Two basic approaches to gene therapy have evolved (1) ex vivo;and (ii) in vivo gene therapy. In ex vivo gene therapy cells are removedfrom a patient or are derived from another source, and while beingcultured are treated in vitro. Generally, a functional replacement geneis introduced into the cell via an appropriate gene deliveryvehicle/method (transfection, transduction, homologous recombination,etc.) and an expression system as needed and then the modified cells areexpanded in culture and returned to the host/patient. These geneticallyreimplanted cells have been shown to express the transfected geneticmaterial in situ.

[0164] In in vivo gene therapy, target cells are not removed from thesubject rather the genetic material to be transferred is introduced intothe cells of the recipient organism in situ, that is within therecipient. In an alternative embodiment, if the host gene is defective,the gene is repaired in situ (Culver, 1998. (Abstract) Antisense DNA &RNA based therapeutics, February 1998, Coronado, Calif.). Thesegenetically altered cells have been shown to express the transfectedgenetic material in situ.

[0165] The gene expression vehicle is capable of delivery/transfer ofheterologous nucleic acid into a host cell. The expression vehicle mayinclude elements to control targeting, expression and transcription ofthe nucleic acid in a cell selective manner as is known in the art. Itshould be noted that often the 5′UTR and/or 3′UTR of the gene may bereplaced by the 5′UTR and/or 3′UTR of the expression vehicle. Therefore,as used herein the expression vehicle may, as needed, not include the5′UTR and/or 3′UTR of the actual gene to be transferred and only includethe specific amino acid coding region.

[0166] The expression vehicle can include a promoter for controllingtranscription of the heterologous material and can be either aconstitutive or inducible promoter to allow selective transcription.Enhancers that may be required to obtain necessary transcription levelscan optionally be included. Enhancers are generally any nontranslatedDNA sequence which works contiguously with the coding sequence (in cis)to change the basal transcription level dictated by the promoter. Theexpression vehicle can also include a selection gene as described hereinbelow.

[0167] Vectors can be introduced into cells or tissues by any one of avariety of known methods within the art. Such methods can be foundgenerally described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor Laboratory, New York 1989, 1992), in Ausubelet al., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. 1989), Chang et al., Somatic Gene Therapy, CRC Press, AnnArbor, Mich. 1995), Vega et al., Gene Targeting, CRC Press, Ann ArborMich. (995), Vectors: A Survey of Molecular Cloning Vectors and TheirUses, Butterworths, Boston Mass. 1988) and Gilboa et al. (Biotechniques4 (6): 504-512, 1986) and include, for example, stable or transienttransfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. No. 4,866,042 forvectors involving the central nervous system and also U.S. Pat. Nos.5,464,764 and 5,487,992 for positive-negative selection methods.

[0168] Introduction of nucleic acids by infection offers severaladvantages over the other listed methods. Higher efficiency can beobtained due to their infectious nature. Moreover, viruses are veryspecialized and typically infect and propagate in specific cell types.Thus, their natural specificity can be used to target the vectors tospecific cell types in vivo or within a tissue or mixed culture ofcells. Viral vectors can also be modified with specific receptors orligands to alter target specificity through receptor mediated events.

[0169] A specific example of DNA viral vector introducing and expressingrecombination sequences is the adenovirus-derived vector Adenop53TK.This vector expresses a herpes virus thymidine kinase (TK) gene foreither positive or negative selection and an expression cassette fordesired recombinant sequences. This vector can be used to infect cellsthat have an adenovirus receptor which includes most tissues ofepithelial origin as well as others. This vector as well as others thatexhibit similar desired functions can be used to treat a mixedpopulation of cells and can include, for example, in vitro or ex vivoculture of cells, a tissue or a human subject.

[0170] Features that limit expression to particular cell types can alsobe included. Such features include, for example, promoter and regulatoryelements that are specific for the desired cell type.

[0171] In addition, recombinant viral vectors are useful for in vivoexpression of a desired nucleic acid because they offer advantages suchas lateral infection and targeting specificity. Lateral infection isinherent in the life cycle of, for example, retroviruses, and is theprocess by which a single infected cell produces many progeny virionsthat bud off and infect neighboring cells. The result is that a largearea becomes rapidly infected, most of which was not initially infectedby the original viral particles. This is in contrast to vertical-typesof infections, in which the infectious agent spreads only throughdaughter progeny. Viral vectors can also be produced that are unable tospread laterally. This characteristic can be useful if the desiredpurpose is to introduce a specified gene into only a localized number oftargeted cells.

[0172] As described above, viruses are very specialized infectiousagents that have evolved, in many cases, to elude host defensemechanisms. Typically, viruses infect and propagate in specific celltypes. The targeting specificity of viral vectors utilizes its naturalspecificity to specifically target predetermined cell types and therebyintroduce a recombinant gene into the infected cell. The vector to beused in the methods and compositions of the invention will depend ondesired cell type to be targeted and will be known to those skilled inthe art.

[0173] Retroviral vectors can be constructed to function either asinfectious particles or to undergo only a single initial round ofinfection. In the former case, the genome of the virus is modified sothat it maintains all the necessary genes, regulatory sequences andpackaging signals to synthesize new viral proteins and RNA. Once thesemolecules are synthesized, the host cell packages the RNA into new viralparticles which are capable of undergoing further rounds of infection.The vector's genome is also engineered to encode and express the desiredrecombinant gene. In the case of non-infectious viral vectors, thevector genome is usually mutated to destroy the viral packaging signalthat is required to encapsulate the RNA into viral particles. Withoutsuch a signal, any particles that are formed will not contain a genomeand therefore cannot proceed through subsequent rounds of infection. Thespecific type of vector will depend upon the intended application. Theactual vectors are also known and readily available within the art orcan be constructed by one skilled in the art using well-knownmethodology.

[0174] The recombinant vector can be administered in several ways. Ifviral vectors are used, for example, the procedure can take advantage oftheir target specificity and consequently, do not have to beadministered locally at the diseased site. However, local administrationcan provide a quicker and more effective treatment.

[0175] Procedures for in vivo and ex vivo cell transformation includinghomologous recombination employed in knock-in procedures are set forthin, for example, U.S. Pat. Nos. 5,487,992, 5,464,764, 5,387,742,5,360,735, 5,347,075, 5,298,422, 5,288,846, 5,221,778, 5,175,385,5,175,384, 5,175,383, 4,736,866 as well as Burke and Olson, Methods inEnzymology, 194:251-270 1991); Capecchi, Science 244:1288-1292 1989);Davies et al., Nucleic Acids Research, 20 (11) 2693-2698 1992);Dickinson et al., Human Molecular Genetics, 2(8): 1299-1302 1993); Duffand Lincoln, “Insertion of a pathogenic mutation into a yeast artificialchromosome containing the human APP gene and expression in ES cells”,Research Advances in Alzheimer's Disease and Related Disorders, 1995;Huxley et al., Genomics, 9:742-750 1991); Jakobovits et al., Nature,362:255-261 1993); Lamb et al., Nature Genetics, 5: 22-29 1993); Pearsonand Choi, Proc. Natl. Acad. Sci. USA 1993). 90:10578-82; Rothstein,Methods in Enzymology, 194:281-301 1991); Schedl et al., Nature, 362:258-261 1993); Strauss et al., Science, 259:1904-1907 1993). Further,Patent Applications WO 94/23049, WO93/14200, WO 94/06908, WO 94/28123also provide information.

[0176] Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

[0177] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non limitingfashion.

[0178] Generally, the nomenclature used herein and the laboratoryprocedures utilized in the present invention include molecular,biochemical, microbiological and recombinant DNA techniques. Suchtechniques are thoroughly explained in the literature. See, for example,“Molecular Cloning: A Laboratory Manual” Sambrook et al., (1989);“Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M.,ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”,John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and Experimental Methods

[0179] Materials: Tissue culture media and serum were purchased fromBiological Industries (Beit HaEmek, Israel). Enhanced ChemicalLuminescence (ECL) was performed with a kit purchased from BioRad(Israel). Monoclonal anti p-tyr antibody was purchased from UpstateBiotechnology Inc. (Lake Placid, N.Y., USA). Polyclonal and monoclonalantibodies to PKC isoforms were purchased from Santa Cruz (Calif., USA)and Transduction Laboratories (Lexington, Ky.). The α6 rat antimouse mAb(GoH3) was purchased from Pharmingen (San Diego, Calif.). The antibody6844 for the α6A cytoplasmic domain was a gift from Dr. V. Quaranta(Scripps Research Institute, La Jolla, Calif.). The rat mAb directedagainst the extracellular domain of mouse β4 (346-11A) was a gift fromDr. S. J. Kennel (Oak Ridge National Laboratory, Oak Ridge, Term.). RatmAB to phosphotyrosine was purchased from Sigma (St. Louis, Mo.) andrabbit anti phosphoserine was purchased from Zymed (San Francisco,Calif.). Horseradish peroxidase-anti-rabbit and anti-mouse IgG wereobtained from Bio-Rad (Israel). Leupeptin, aprotinin, PMSF, DTT,Na-orthovanadate, and pepstatin were purchased from Sigma Chemicals (St.Louis, Mo.). Insulin (humulinR-recombinant human insulin) was purchasedfrom Eli Lilly France SA (Fergersheim, France). IGF1 was a gift fromCytolab (Israel). Keratin 14 antibody was purchased from Babco-Convance(Richmond, Calif.) BDGF-BB was purchased from R&D systems (Minneapolis)and PKCα pseudosubstrate myristolated was purchased from Calbinochem(San Diego, Calif.).

[0180] Isolation and culture of murine keratinocytes: Primarykeratinocytes were isolated from newborn skin as previously described(18). Keratinocytes were cultured in Eagle's Minimal Essential Medium(EMEM) containing 8% Chelex (Chelex-100, BioRad) treated fetal bovineserum. To maintain a proliferative basal cell phenotype, the final Ca²⁺concentration was adjusted to 0.05 mM. Experiments were performed fiveto seven days after plating.

[0181] Preparation of cell extracts and Western blot analysis: For crudemembrane fractions, whole cell lysates were prepared by scraping cellsinto PBS containing 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/mlpepstatin, 1 mM PMSF, 10 mM EDTA, 200 μM NaVO₄ and 10 mM NaF. Afterhomogenization and 4 freeze/thaw cycles, lysates were spun down at 4° C.for 20 minutes in a microcentrifuge at maximal speed. The supernatantcontaining the soluble cytosol protein fraction was transferred toanother tube. The pellet was resuspended in 250 μl PBS containing 1%Triton X-100 with protease and phosphatase inhibitors, incubated for 30minutes at 4° C. and spun down in a microcentrifuge at maximal speed at4° C. The supernatant contains the membrane fraction. Proteinconcentrations were measured using a modified Lowery assay (Bio-Rad DCProtein Assay Kit). Western blot analysis of cellular protein fractionswas carried out as described (6).

[0182] Preparation of cell lysates for immunoprecipitation: Culturedishes containing keratinocytes were washed with Ca²⁺/Mg²⁺-free PBS.Cells were mechanically detached in RIPA buffer (50 mM Tris•HCl pH 7.4;150 mM NaCl; 1 mM EDTA; 10 mM NaF; 1% Triton x100; 0.1% SDS, 1% Nadeoxycholate) containing a cocktail of protease and phosphataseinhibitors (20 μg/ml leupeptin; 10 μg/ml aprotinin; 0.1 mM PMSF; 1 mMDTT; 200 μM orthovanadate; 2 μg/ml pepstatin). The preparation wascentrifuged in a microcentrifuge at maximal speed for 20 minutes at 4°C. The supernatant was used for immunoprecipitation.

[0183] Immunoprecipitation: The lysate was precleared by mixing 300 μgof cell lysate with 25 μl of Protein A/G Sepharose (Santa Cruz, Calif.,USA), and the suspension was rotated continuously for 30 minutes at 4°C. The preparation was then centrifuged at maximal speed at 4° C. for 10minutes, and 30 μl of A/G Sepharose was added to the supernatant alongwith specific polyclonal or monoclonal antibodies to the individualantigens (dilution 1:100). The samples were rotated overnight at 4° C.The suspension was then centrifuged at maximal speed for 10 minutes at4° C., and the pellet was washed with RIPA buffer. The suspension wasagain centrifuged at 15,000×g (4° C. for 10 minutes) and washed fourtimes in TBST. Sample buffer (0.5 M Tris-HCl pH 6.8; 10% SDS; 10%glycerol; 4% 2-beta-mercaptoethanol; 0.05% bromophenol blue) was addedand the samples were boiled for 5 minutes and then subjected toSDS-PAGE.

[0184] Attachment assays: Twenty four well petri plates (Greiner) werecoated (250 μl/well) with 20 μg/ml of matrix proteins in PBS for 1 hourat 37° C. Following incubation, plates were washed and incubated with0.1% BSA for 30 minutes at room temperature to block nonspecificbinding. Keratinocytes cultures were trypsinized briefly with 0.25%trypsin and following detachment, cells were resuspended andkeratinocytes (1×10⁶) added to the coated wells and incubated for 1 hourat 37° C. Nonadherent cells were removed, the wells were rinsed twicewith PBS and the remaining cells were extracted in 1 M NaOH. Cell countwas determined by protein concentrations using a modified Lowery assay(Bio-Rad DC Protein Assay Kit). Results were calculated by percentagerelative to untreated controls.

[0185] Immunofluorescence: Primary keratinocytes were plated on laminin5 coated glass slides. Two days old keratinocytes were infected with PKCadenovirus for one hour, washed twice with PBS and maintained in culturein low Ca²⁺ EMEM. Twenty four hours post infection, cells were fixed in4% paraformaldehyde for 30 minutes followed by permeabilization with0.2% Triton for 5 minutes. For analysis, control and PKC infectedkeratinocytes were rinsed with PBS and incubated overnight at 4° C. withPKC antibodies (Santa Cruz) diluted in 1% BSA in PBS. After incubation,slides were washed twice for 10 minutes with PBS and incubated withbiotinylated secondary anti rabbit antibody for 20 minutes, washed twicein PBS and incubated with Strepavidin-FITC for 20 minutes. For analysisof α6β4 staining, glass slides were treated with 0.2% triton X-100 for 5minutes on ice followed by 5 minutes fixation in methanol. The slideswere incubated with anti α6 or anti β4 antibodies overnight followed byincubation with biotinylated secondary anti rat antibody, respectively,for 20 minutes, washed twice in PBS and incubated with Strepavidin-FITCfor 20 minutes. Following two washes in PBS, slides were mounted withglycerol buffer containing 1% of p-phenylenediamine (Sigma) andfluorescence examined by laser scanning confocal imaging microscopy(MRC1024, Bio-Rad, UK).

[0186] Adenovirus constructs: The recombinant adenovirus vectors wereconstructed as previously described (19). The dominant negative mutantsof mouse PKCs were generated by substitution of the lysine residue atthe ATP binding site with alanine. The mutant cDNA was cut from SRDexpression vector with EcoR I and ligated into the pAxCA1w cosmidcassette to construct the Ax vector. The dominant negative activity ofthe genes was demonstrated by the abrogation of its autophosphorylationactivity.

[0187] Transduction of keratinocytes with PKC isoform genes: The culturemedium was aspirated and keratinocyte cultures were infected with theviral supernatant containing PKC recombinant adenoviruses for one hour.The cultures were then washed twice with MEM and re-fed. Ten hourspost-infection cells were transferred to serum-free low Ca²⁺-containingMEM for 24 hours. Keratinocytes from control and insulin-treated orIGF1-treated cultures were used for proliferation assays, ⁸⁶Rb uptake,or extracted and fractionated into cytosol and membrane fractions forimmunoprecipitation, immunofluorescence and Western blotting asdescribed.

[0188] PKC activity: Specific PKC activity was determined in freshlyprepared immunoprecipitates from keratinocyte cultures followingappropriate treatments. These lysates were prepared in RIPA bufferwithout NaF. Activity was measured with the use of the SignaTECT ProteinKinase C Assay System (Promega, Madison, Wis., USA) according to themanufacturer's instructions. PKCα pseudosubstrate was used as thesubstrate in these studies.

[0189] Cell proliferation: Cell proliferation was measured by[³H]thymidine incorporation in 24 well plates. Cells were pulsed with[³H]thymidine (1 μCi/ml) overnight. After incubation, cells were washedfive times with PBS and 5% TCA was added into each well for 30 minutes.The solution was removed and cells were solubilized in 1% Triton X-100.The labeled thymidine incorporated into cells was counted in a ³H-windowof a Tricarb liquid scintillation counter.

[0190] Na⁺/K⁺ pump activity: Na⁺/K⁺ pump activity was determined by themeasurements of ouabain-sensitive uptake of ⁸⁶Rb by whole cells in 1 mlof K⁺-free PBS containing 2 mM RbCl and 2.5 μCi of ⁸⁶Rb. Rb uptake wasterminated after 15 minutes by aspiration of the medium, after which thecells were rinsed rapidly four times in cold 4° C. K⁺-free PBS andsolubilized in 1% Triton X-100. The cells from the dish were added to 3ml H₂O in a scintillation vial. Samples were counted in a ³H-window of aTricarb liquid scintillation counter. Rb-uptake specifically related toNa⁺/K⁺ pump activity was determined by subtraction of the cpmaccumulated in the presence of 10⁻⁴ M ouabain from the uptake determinedin the absence of the inhibitor.

[0191] PKC immunokinase assay: Purified and standardized PKC isozymeswere kindly supplied by Dr. P. Blumberg (NCI, NIH, U.S.) and Dr.Marcello G. Kazanietz (University of Pennsylvania, School of Medicine).Primary keratinocytes were harvested in 500 μl 1% Triton Lysis Buffer(1% Triton-X 100, 10 μg/ml aprotinin and leupeptin, 2 μg/ml pepstatin, 1mM PMSF, 1 mM EDTA, 200 μM Na₂VO₄, 10 mM NaF in 1×PBS). Lysates wereincubated at 4° C. for 30 minutes, and spun at 16,000×g for 30 minutesat 4° C. Supernatants were transferred to a fresh tube.Immunoprecipitation of cell lysates was carried out overnight at 4° C.with 5 μg/sample anti-α6/GoH3 (PharMingen) and 30 μl/sample of proteinA/G-Plus agarose slurry (Santa Cruz). Beads were washed once with RIPAbuffer and twice with 50 mM Tris/HCl pH 7.5. 35 μl of reaction buffer (1mM CaCl₂, 20 mM MgCl2, 50 mM Tris•HCl pH 7.5) was added to each assay.To each assay, 5.5 μl/assay of a suspension of phospholipid vesiclescontaining either DMSO or 10 mM TPA was added to the slurry togetherwith a standardized amount of specific PKC isozyme. The reaction wasinitiated by adding 10 μl/assay 125 mM ATP (1.25 μCi/assay [γ-32P] ATP,Amersham) and allowed to continue for 10 minutes at 30° C. The beadswere then washed twice with RIPA buffer. 30 μl/sample protein loadingdye (3× Laemmli, 5% SDS) was added and the samples were boiled for 5minutes in a water bath. Proteins were separated by SDS-PAGE on a 8.5%gel, transferred onto Protran membranes (Schleicher & Schuell) andvisualized by autoradiography. Phosphorylation of histones andphosphorylation of PKC substrate peptide were used as controls for PKCactivity.

EXPERIMENTAL RESULTS Example 1 Effective Over-Expression of PKC IsoformsUtilizing Recombinant Adenovirus Vectors

[0192] By utilizing a recombinant β-galactosidase adenovirus a highinfection rate was achieved with more then 90% of the culturedkeratinocyte population expressing the recombinant protein. Therecombinant β-galactosidase adenovirus infection did not affect cellviability or cell growth. Furthermore, β-galactosidase expression wassustained for up to two weeks of culture and was used as a controlinfection in following experiments. The efficiency of recombinant PKCadenovirus constructs to induce protein expression and be activatedproperly in mouse keratinocyte cultures was examined. As seen by Westernblotting in FIG. 1, 24 hours following a 1 hour infection withrecombinant PKC adenovirus constructs, a dramatic increase in specificPKC protein expression was observed five to ten fold above theendogenous expression levels of the specific isoforms. Recombinantprotein could be detected in infected keratinocyte cultures as early as6 hours following infection and peak expression was obtained by 24hours. Protein expression was sustained throughout the culture period(up to fourteen days).

Example 2 Over-Expressed PKC Isoforms are Activated by PKC Activators

[0193] Recombinant proteins of the PKC isoforms responded typically toPKC activators. As seen in FIG. 2, treatment with bryostatin 1 inducedtranslocation of PKCα and δ proteins to the membrane fraction, with alesser effect on PKCη and ζ isoforms, similarly to results obtained withthe endogenous isoforms and as expected from their cofactorrequirements.

Example 3 Over-Expressed PKC Isoforms are Active in Their Native Form

[0194] As early as 18 hours following infection, PKC kinase assaysrevealed that immunoprecipitates of distinct PKC isoforms wereenzymatically active without further need of stimulation by PKCactivators (FIG. 3).

Example 4 Over-Expression of Specific PKC Isoforms Induces DistinctMorphological Changes in Primary Keratinocytes

[0195] Each of the PKC adenovirus constructs employed induced a specificmorphological change in primary keratinocytes (FIG. 4). Uninfectedprimary mouse keratinocyte cultures and β-galactosidase infected cellspresented a cubidal morphology typical to the proliferative basal cellcharacteristics in culture. Regardless of isoform specificity all PKCover-expressing keratinocytes showed morphological changes typical toPKC activation including cell elongation and the appearance of neuronallike projections. However, each one of the PKC isoforms had acharacteristic effect on keratinocyte morphology. PKCα infection inducedstratification of keratinocytes, with a typical flattened morphology. Incontrast, PKCη appeared as condensed clones of cells, presentingmorphological characteristics of basal cells proliferating at promptrate (FIG. 4). Two of the isoforms appeared to effect cell matrix aswell as cell-cell associations. 18-48 hours following PKCδ infection,cells appeared elongated and extended with neuronal like projections.This was followed by gradual cell loss off the culture dish whichoccurred progressively in the course of the culture period.Over-expressing PKCζ keratinocytes appeared as rounded keratinocyteclusters, which were attached loosely to the culture dish and weregradually lost several days following infection.

Example 5 Distinct Localization of Over-Expressed PKC Isoforms inInfected Primary Keratinocytes

[0196] The distinct morphological changes were associated with distinctcellular localization as characterized by immunofluorescence analysis.In proliferating keratinocytes, PKCα, PKCδ and PKCζ were expressed inthe cytoplasm as well as in the plasma membrane. Similarly to endogenousprotein expression, PKCη isoform was localized to the keratinocytes'perinuclear region (FIG. 5). A dynamic change in distribution wasassociated with PKCδ and PKCζ, where succeeding cell detachment PKCisoform expression was predominantly localized to the cell membrane(FIG. 5).

Example 6 Regulation of α6β4 Expression by PKC Isoforms ExperimentalResults

[0197] The ability of specific PKC isoforms to regulate proteins whichare characteristic of the basal phenotype of the proliferative basallayer was examined. As down regulation of α6β4 integrin is one of theearly events taking place during keratinocyte differentiation, theability of the various PKC isoforms to regulate expression of the α6β4integrin, an integrin which is specifically localized to thehemidesmosomes of the basal layer was assessed. As can be seen in theimmunoblot presented in FIG. 6, only PKCδ and PKCζ isoforms were able todown regulate α6β4 expression, in comparison to α6β4 integrin subunitslevels in control keratinocytes. At the same time, α3 or β1 integrinsubunits levels were not reduced. In contrast, consistently,over-expression of PKCα isoform resulted in increased α6β4 level two tothree fold above control expression (FIG. 6). Over-expression of PKCηdid not effect α6β4 protein expression. Several characteristics areassociated with commitment of cells to differentiation and which followthe down regulation of the α6β4 protein including decrease in theproliferation rate, new keratin synthesis, cellular detachment and lossof attachment to basement membrane components. No changes in keratinexpression were observed by over-expression of the different PKCisoforms. This included expression of K5 and K14, which arecharacteristic of the basal proliferating keratinocytes and K1 and K10,which are characteristic of the early stages of spinous differentiation.In addition, when proliferation rate was analyzed by ³H-thymidineincorporation there was no correlation between the loss of α6β4expression and proliferation potential.

Example 7 Over-Expressed PKCη and PKCδ Induce KeratinocytesProliferation In Vitro

[0198] Over-expression of PKCη and PKCδ significantly inducedkeratinocyte proliferation five and two fold above control levelsrespectively (FIG. 7). PKCζ and PKCα did not affect cell proliferation.

Example 8 Over-Expressed PKC δ and ζ Induce Keratinocytes Detachment InVitro

[0199] The adhesion properties of PKCδ and ζ over-expressingkeratinocytes was studied. In comparison to control keratinocytes nochange in adhesion potential to specific matrix proteins includinglaminin1, laminin 5, fibronectin and collagen, was observed (data notpresented). However, in cells over-expressing PKCδ and PKCζ isoforms,loss of cell contact with the culture dish was associated with gradualkeratinocyte detachment from the culture dish (FIG. 4).

Example 9 PKC Isoforms Over-Expression Effects on HemidesmosomalLocalization of α6β4 Integrin

[0200] As α6β4 expression is essential for the formation of thehemidesmosomal adhesion complex, the association of α6β4 down regulationand cell detachment with α6β4 localization to the hemidesmosome wasexamined. FIG. 8 presents immunofluorescent analysis of α6β4 associationwith the hemidesmosomal complexes. As seen in FIG. 8, in comparison tocontrol infected keratinocytes, up regulation of α6β4 integrinexpression in over-expressing PKCα keratinocytes (FIG. 6) is associatedwith increased integration of α6β4 to the hemidesmosomal complexes.Cells over-expressing PKCη also induced association of α6β4 integrinwith the hemidesmosomal complexes, although less than observed inover-expressing PKCα cells. As expected, the significant down regulationof α6β4 integrin in PKCδ and PKCζ over-expressing keratinocytes wasfound to be associated with decreased integration of α6β4 with thecells' hemidesmosomal complexes (FIG. 8). These results suggest thatα6β4 integrin plays an important role in cell-matrix association andkeratinocytes encoring to the underlying basement membrane. Furthermore,PKCδ and ζ mediated α6β4 down regulation, initiate keratinocyte celldetachment in a pathway distinct from the keratinocyte differentiationprocesses. Finally, in order to link PKC mediated α6β4 down regulation,decrease hemidesmosomal α6β4 integration and specific morphologicalchanges to keratinocyte detachment, the changes in the amount ofattached and detached cells over-expressing the different PKC isoformsduring the culture period were followed. In FIG. 9, attached cells werecounted in cultures 24 and 48 hours following PKC adenoviral infection.As can be clearly observed, both PKCδ and PKCζ induced cell loss invitro. In parallel, the loss of cells in culture was correlated with theincrease in cells floating in the overlaying medium. These resultsindicate that PKCδ and PKCζ are important for control of the detachmentstep associated with the early stages of cell differentiation.

Example 10 PKCη Differentially Regulate Keratinocyte Proliferation andDifferentiation Under Physiological Settings

[0201] As clearly shown in FIG. 7, cells over-expressing PKCη isoformproliferate at an accelerated rate, five to seven times above controluninfected cells, and consistently higher then keratinocyte culturesover-expressing other PKC isoforms. However, the induction ofproliferation was dependent on the differentiation state of thekeratinocytes as determined by regulating the Ca²⁺ concentrations in themedium. In proliferating keratinocytes maintained under low Ca²⁺concentrations (0.05 mM) endogenous PKCη was localized to theperinuclear region of majority of the proliferating cells (FIG. 10).Under these conditions, PKCη over-expression induced a dramatic increasein keratinocyte proliferation (FIG. 11). However, when keratinocyteswere differentiated by elevating the Ca²⁺ concentrations to 0.12 mM,over-expression of PKCη did not induce proliferation but furtherstimulated keratinocyte differentiation. These results suggest thatover-expressed PKCη induces proliferation only in physiologicallyproliferating cells but does not interfere with cellulardifferentiation. Divergence in regulation of PKCη expression was alsoseen in vivo. PKCη expression in actively proliferating skin as well asneuronal cells of the embryo was identified while in the mature adultbrain no PKCη was observed and in the epidermis PKCη was localized tothe granular layer in skin.

Example 11 PKCη and DNPKCη Over-Expression Specifically Regulates PKCLocalization and Cellular Morphology

[0202] To further corroborate the results which support a positive rolefor PKCη in both states of proliferation or differentiation inkeratinocytes, the effects of a kinase inactive dominant negativeadenovirus PKCη construct were analyzed by studying the effect ofinfection in proliferating and differentiating keratinocytes. As seen inFIG. 12 adenoviral infection of both PKCη and DNPKCη were efficient inboth the proliferative and differentiative states. As predicted, inproliferating keratinocytes DNPKCη induced keratinocyte differentiationwith a dramatic change in cell morphology including flattening of thecells, loss of cell-cell boundaries similarly to the morphologicalchanges associated with Ca²⁺ induced differentiation (FIGS. 12A-B).Furthermore, these changes were associated with shut off of keratinocyteproliferation (FIG. 11) and a dramatic induction of differentiationmarkers including keratin 1, keratin 10, loricrin and Filagrin, whichwere elevated to similar levels presented in normal skin in vivo (FIGS.13A-B). At the same time, upon initiation of the differentiationprogram, over-expression of DNPKCη did not abrogate Ca²⁺ induceddifferentiation. These results suggest that PKCη and DNPKCη can be usedfor differentially regulating keratinocyte proliferation anddifferentiation under physiological settings.

Example 12 In Vivo Experiments

[0203] In order to test the ability of PKCη to differentially regulatecell proliferation and differentiation in vivo, the ability of PKCη toinduce healing of full incisional wounds created on the back of nudemice was assessed. The ability of the keratinocytes to express theexogenous recombinant protein was verified by utilizing a control β-galadenovirus. As can be seen in FIG. 14, two weeks after infection, β-galexpression is maintained in vitro keratinocytes as well as in vivo skin.Interestingly, when the wound healing process was examined in mice afterlocal infection with control, PKCα and PKCη adenovirus constructs, onlyPKCη induced the formation of granulation tissue as early as four daysfollowing topical infection. This included also the organized formationof muscle, fat and dermal layers. At the same time in control and PKCαinfected skins, condensed granulation tissue was not noticed and noclosure of the wound was observed (FIG. 14). Therefore, PKCη can beconsidered as a primary candidate in regulating proliferation anddifferentiation of skin in the induction of wound healing processes.

Example 13 Insulin Specifically Induces Translocation of PKCδ inProliferating Keratinocytes

[0204] Two PKC isoforms expressed in skin were found to affectkeratinocyte proliferation: PKCη and PKCδ. In order to try and identifythe endogenous factors, which activate specific PKC isoforms regulatingskin proliferation, the ability of several growth factors which areknown to promote keratinocyte proliferation including: EGF, KGF,insulin, PDGF and IGF1 to activate specific PKC isoforms in a growthdependent manner was assessed. PKC isoforms α, δ, ε, η and ζ areexpressed in the skin. As activation of PKC isoforms is associated withtheir translocation to membrane fractions, the effects of these growthfactors on the translocation of the various PKC isoforms from cytosol tothe membrane were examined. As seen in FIG. 15, as early as 5 minutesfollowing stimulation, insulin specifically induced translocation ofPKCδ from the cytoplasm to the membranal fractions. Membrane expressionof PKCδ was maintained for several hours following insulin stimulation.In contrast, IGF1 reduced PKCδ expression in the membrane and increasedits relative level of expression in the cytoplasm fraction. No othergrowth factor significantly affected PKCδ translocation andlocalization. No change in distribution of the other PKC isoforms wasseen following stimulation by any of the growth factors including IGF1and insulin.

Example 14 Insulin Specifically Induces Activation of PKCδ inProliferating Keratinocytes

[0205] In order to determine whether the translocation of PKCδ issufficient for activation, kinase activity of PKC immunoprecipitatesfrom the cytoplasm and membrane fractions of insulin and IGF1 treatedkeratinocytes was measured. As shown in FIG. 16, insulin but not IGF1increased activity of PKCδ in the membrane fraction. No elevation inPKCα activity was observed in the cytoplasm fraction. Theinsulin-induced activation was specific for PKCδ and no activation ofPKCs α, ε, η or ζ was observed for up to 30 minutes following insulinstimulation. Altogether, these results suggest selective stimulation byinsulin but not IGF1 of PKCδ activation.

Example 15 Insulin and IGF1 have an Additive Effect on KeratinocyteProliferation

[0206] In order to analyze if the specific activation of PKCδ signifiesspecific insulin induced mitogenic pathway in keratinocytes themitogenic effects of both insulin and IGF1 were examined by studyingtheir ability to induce keratinocyte proliferation as measured bythymidine incorporation. As shown in FIG. 17A, both insulin and IGF1stimulated thymidine incorporation in a dose dependent manner withmaximal induction achieved at 10⁻⁷ and 10⁻⁸ M, respectively. At eachconcentration, the maximal stimulation by IGF1 was greater than that byinsulin. Interestingly, at all concentrations, when both hormones weregiven together, the mitogenic effects were additive (FIG. 17B). Theseresults suggest that insulin regulates keratinocyte proliferationthrough a distinct pathway independent of IGF1 induced keratinocyteproliferation.

Example 16 The Association Between Insulin-Induced PKCδ Activation andInsulin-Induced Keratinocyte Proliferation

[0207] In order to directly study the association betweeninsulin-induced PKCδ activation and insulin-induced keratinocyteproliferation, recombinant PKC adenovirus constructs were used toover-express both wild type PKCδ (WTPKCδ) as well as a kinase-inactivedominant negative mutant of PKC, which abrogates the endogenous PKCδactivity (DNPKCδ). The effects of over-expression of WTPKCδ and DNPKCδon insulin-induced keratinocyte proliferation were examined. Bothconstructs, as well as a PKCα construct, were efficiently expressed inkeratinocytes (FIG. 18A). Furthermore, infection with PKCδ and PKCαinduced isoform-specific PKC activity several fold above control levels(FIG. 18B). As expected, over-expression of DNPKCδ did not induce PKCactivity. As can be seen in FIG. 19A, insulin treatment of untransfectedcells or over-expression of WTPKCδ without insulin treatment, increasedthymidine incorporation to approximately identical levels, two to threefold over untreated cells, or cells transduced with PKCα. Moreover,addition of insulin to cells already over-expressing WTPKCδ did notcause any additional increase in thymidine incorporation. IGF1 increasedthymidine uptake similarly in both non-infected cells and in cellsover-expressing WTPKCδ and PKCα (FIG. 19A). The direct involvement ofPKCδ in insulin induced proliferation was further proven by abrogatingPKCδ activity. As seen in FIG. 19B, basal thymidine incorporation incells over-expressing the dominant negative PKCδ was slightly, butsignificantly, lower than that in non-infected cells. Over-expression ofDNPKCδ completely eliminated insulin-induced proliferation but did notaffect IGF1-induced proliferation. Moreover, the additive effects ofinsulin and IGF1 was reduced to that of IGF1 alone.

Example 17 Specificity of PKCδ Activation to the Insulin-MediatedPathway

[0208] The specificity of PKCδ activation to the insulin-mediatedpathway was analyzed by investigating the effects of PKCδ and DNPKCδ onthe mitogenic response to a variety of growth factors including: IGF 1,EGF, KGF, ECGF and PDGF. As seen in FIG. 20, the over-expression ofDNPKCδ selectively eliminated the proliferative effects induced byinsulin but did not block those of any of the other growth factorstested. However, the over-expression of PKCδ mimicked insulin inducedproliferation and did not affect IGF1 induced proliferation. Theproliferation induced by stimulation with EGF and KGF was increased(FIG. 21). These data indicate that PKCδ activation by insulin, mediatesproliferation of keratinocytes through a pathway involving PKCδ and thatthis pathway is upstream of EGF and KGF signaling, two major growthfactors known to regulate keratinocyte proliferation. Overall, insulinwas found to be a specific regulator of PKCδ activity, which could be aspecific candidate in regulating keratinocyte proliferation induced byinsulin, EGF and KGF.

Example 18 Insulin Induced PKCδ Activity and Keratinocyte Proliferationis Mediated by STAT3 Transcriptional Activation

[0209] The role of PKCδ in insulin signaling was further characterizedand found to involve induction of transcriptional activation mediated bySTAT3. As senn in FIG. 23, in primary keratinocytes, PKCδ was shown tospecifically associate with STAT3. Following insulin stimulation, PKCδis activated and in turn phosphorylates and activates STAT3 (FIG. 24).Moreover, abrogating PKCδ activity by a pharmacological inhibitor(rottlerin) inhibits activaiton as well as nuclear translocation ofSTAT3. Furthermore, as seen in FIG. 25, overexpression of STAT3 inducesa similar proliferation as that induced by insulin and by overexpressionof PKCδ and abrogation of PKCδ activity by overexpression of a dominantnegative PKCδ mutant abolishes the ability of STAT3 to inducekeraitnocyte proliferation. Overall these results suggest that insulinand PKCδ play a role in transcriptional activation associated withkeratinoycte proliferation.

Example 19 PKCδ and PKCζ are Essential to the Wound Healing Process InVivo

[0210] The importance of PKC isoforms in the wound healing process invivo was established utilizing isoform specific PKC null mice. As seenin FIGS. 22A-B, when fill thickness wounds were created on the back ofPKCδ, PKCζ, PKCα null mice (knock-out, KO) and their wild typelittermates, delayed wound healing was observed in PKCδ and PKCζ but notPKCα null mice. This data indicates that even in the absence of diabeticbackground, specific PKC isoforms are essential for the wound healingprocess in skin.

Example 20 Single vs. Multiple Applications of Insulin for Wound HealingIn Vivo

[0211] Wounds were effected on the back of 8-10 week old C57BL mice byincision and were treated as follows: (i) insulin 0.1 μM applied dailyfor 7 days; (ii) insulin 1 μM applied daily for 7 days (iii) insulin 10μM applied daily for 7 days; (iv) insulin 1 μM applied once 4 days afterwounding; and (v) vehicle (PBS) control applied daily for 7 days. Allmice were sacrificed seven days after wounding and their open woundareas were measured. As seen in FIG. 26, a daily treatment of insulin at1 μM concentration was significantly more effective than dailytreatments of insulin at a lower (0.1 μM) or a higher (10 μM)concentration. Surprisingly, the treatment of a single application ofinsulin at 1 μM concentration was substantially more effective than thetreatment of seven repeat daily applications of insulin at the sameconcentration.

[0212] Since the observed wounds were covered with a scar tissue it wasdifficult to correctly assess the actual closure of the wound and theformation of reconstructed epidermis. Therefore the effects of insulinon epidermal and dermal closure of wounds tissue were determined byhistological parameters. Epidermal closure of wounds was determined bystaining wound sections with Keratin 14 antibody (K14, Babco-Convance,Richmond, Calif., USA) which highlighted the formation of basal cells atthe wound gap. Dermal closure of wounds was considered positive if bothwound sides the dermis could be observed in a single field observedunder a light microscope at ×10 magnification.

[0213] As seen in FIG. 27, all insulin treatments effectively promotedepidermal and dermal closure. Similarly to the results shown in FIG. 26,a daily treatment of insulin at 1 μM concentration was significantlymore effective than a daily treatment of insulin at 0.1 μM, or 10 μMconcentrations. In addition, a single application of insulin at 1 μMconcentration was substantially more effective than of seven repeatdaily applications of insulin at the same concentration.

[0214] Hence, these results clearly substantiate the therapeuticefficacy of insulin on wound healing in vivo as determined bymorphological as well as histological parameters. The resultssurprisingly show that determining the optimal number and/or frequencyof applications of insulin is a critical step for treating woundsproperly.

Example 21 Combining Insulin and Platelet-Derived Growth Factor(PDGF-BB) for Wound Healing In Vivo

[0215] Wounds were effected on the back of 8-10 week old C57BL mice byincision and were treated 4 days after wounding as follows: (i) vehicle(PBS) control; (ii) insulin 1 μM (iii) PDGF-BB 10 μM (R&D Systems,Minneapolis, USA); and (iv) insulin 1 μM+PDGF-BB 10 μM. Three days aftertreatment all mice were sacrificed and the treated wounds werehistologically analyzed for epidermal and dermal closure such asdescribed in Example 20 above.

[0216] As seen in FIG. 28 a treatment with either insulin or PDGF-BBalone was partially effective on epidermal closure (30-40% increase overcontrol) and on dermal closure (10-20% increase over control). However,the treatment of insulin and PDGF-BB combined resulted in substantiallyhigher epidermal closure (ca. 80% over control) as well as dermalclosure (ca. 60%). Thus, the results show that combination of insulinand PDGF-BB affect wound healing in a synergistic manner. The resultsfurther indicate the potential of combining insulin with other growthfactors or transforming factor such as EGF, TGFβ, KGF for therapeutictreatment of wounds.

Example 22 Combining Insulin and PKCα Inhibitor for Wound Healing InVivo

[0217] Wounds were effected on the back of 8-10 week old C57BL mice byincision and were treated daily for 7 days with either vehicle (PBS)control or with 0.67 μM insulin (HO/01; Hunulin, Eli Lilly, USA)combined with a PKCα inhibitor (HO/02; PKCα pseudosubstratemyristolated; Calibiochem, San Diego, Calif., USA). Seven days afterwounding all mice were sacrificed and treated wounds were analyzed forwound closure, epidermal closure, dermal closure, and spatialdifferentiation of epidermal cells. Wound closure was determined bymeasuring the open wound area. Dermal closure of wounds was consideredpositive if both wound sides the dermis could be observed in a singlefield observed under a light microscope at ×10 magnification. Epidermalclosure of wounds was determined by staining wound sections with K14antibody which highlighted the formation of basal cells at the woundgap. Spatial differentiation of epidermal cells was determined bystaining wound sections with K1 antibody which highlighted newly formedepidermal cells.

[0218] As illustrated in FIGS. 28-32 the combined application of insulinand (HO/01) and the PKCα inhibitor (HO/02) substantially promoted woundclosure (FIGS. 29A-B), dermal closure (FIG. 30), epidermal closure (FIG.31), and spatial differentiation of epidermal cells (FIG. 32). As can beseen in FIG. 33, the treatment of insulin HO/01 combined with PKCαinhibitor HO/02 increased wounds epidermal closure from ca. 15 to 70%,increased dermal closure from ca. 15 to 50% and increased spatialdifferentiation of epidermal cells from ca. 15 to 50%, as compared withthe vehicle control, respectively.

[0219] Hence, the results show that a therapeutic treatment of wounds byinsulin combined with a PKCα inhibitor effectively promotes epidermalclosure, dermal closure, spatial differentiation of epidermal cells, andsubsequently wound healing.

Example 23 PKCα Inhibitor Reduces Wounds Inflammation

[0220] Late and severe inflammatory response in wounds may suppress theprocess of healing, thus preventing such inflammation from developmentmay promote the wound healing process. Accordingly, the effect of PKCαinhibitor and insulin on wound inflammation was tested in the followingexperiment.

[0221] Wounds were effected on the back of C57BL mice by incision andwere treated daily for 7 days with: (i) PBS, control; (ii) 1 μM of aPKCα inhibitor (pseudosubstrate myristolated; Calibiochem, USA); (iii) 1μM insulin (Eli Lilly, USA); or a mixture of 1 μM PKCα inhibitor and 1μM insulin. Seven days after wounding all mice were sacrificed and thetreated wounds were observed for inflammation under a microscope. Theresulting incidences of severe inflammation observed in the wound areaare summarized in Table 1 that follows. TABLE 1 Incidence of severeTreatment inflammation in wound (%) PBS Control 60.0 PKCα inhibitor 40.0Insulin 56.0 PKCα inhibitor + insulin 50.0

[0222] The results show that administering the PKCα inhibitor to woundscaused a substantial (33.3%) decrease of severe wound inflammationincidence, as compared to control. Insulin alone was not effective underthe experimental conditions.

[0223] These results indicate that a PKCα inhibitor can be used intherapy to control severe inflammation of wounds. The demonstratedcapacity of PKCα inhibitor to reduce inflammation, coupled with itscapacity to promote epidermal closure, dermal closure and spatialdifferentiation of epidermal cells (see in Example 22 hereinabove),makes it a potentially most effective therapeutic agent for woundhealing.

[0224] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by their name and/or database accession numbersmentioned in this specification are herein incorporated in theirentirety by reference into the specification, to the same extent as ifeach individual publication, patent, patent application or sequence wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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What is claimed is:
 1. A method of inducing or accelerating a healingprocess of a skin wound, the method comprising the step of administeringto the skin wound a therapeutically effective amount of insulin and atleast one additional agent acting in synergy with said insulin to induceor accelerate the healing process of the skin wound.
 2. The method ofclaim 1, wherein said administering is effected by a single application.3. The method of claim 1, wherein said therapeutically effective amountof insulin has an insulin concentration ranging from 0.1 μM to 10 μM. 4.The method of claim 4, wherein said at least one additional agent is aplatelet-derived growth factor.
 5. The method of claim 1, wherein saidat least one additional agent is a PKC-α inhibitor.
 6. The method ofclaim 1, wherein said wound is selected from the group consisting of anulcer, a burn, a laceration and a surgical incision.
 7. The method ofclaim 6, wherein said ulcer is a diabetic ulcer.
 8. The method of claim1, wherein said insulin is recombinant.
 9. The method of claim 1,wherein said insulin is of a natural source.
 10. The method of claim 1,wherein said insulin and at least one additional agent are contained ina pharmaceutical composition adapted for topical application.
 11. Themethod of claim 10, wherein said pharmaceutical composition is selectedfrom the group consisting of an aqueous solution, a gel, a cream, apaste, a lotion, a spray, a suspension, a powder, a dispersion, a salveand an ointment.
 12. The method of claim 10, wherein said pharmaceuticalcomposition includes a solid support.
 13. A method of inducing oraccelerating a healing process of a skin wound, the method comprisingthe step of implanting into the skin wound a therapeutically effectiveamount of insulin secreting cells, so as to induce or accelerate thehealing process of the skin wound.
 14. The method of claim 13, whereinsaid wound is selected from the group consisting of an ulcer, a burn, alaceration and a surgical incision.
 15. The method of claim 14, whereinsaid ulcer is a diabetic ulcer.
 16. The method of claim 13, wherein saidcells are transformed to produce and secrete insulin.
 17. The method ofclaim 16, wherein said cells are transformed by a recombinant PDX1 geneand therefore said cells produce and secrete natural insulin.
 18. Themethod of claim 16, wherein said cells are transformed by a cis-actingelement sequence integrated upstream to an endogenous insulin gene ofsaid cells and therefore said cells produce and secrete natural insulin.19. The method of claim 16, wherein said cells are transformed by arecombinant insulin gene and therefore said cells produce and secreterecombinant insulin.
 20. The method of claim 13, wherein said insulinsecreting cells are capable of forming secretory granules.
 21. Themethod of claim 13, wherein said insulin secreting cells are endocrinecells.
 22. The method of claim 13, wherein said insulin secreting cellsare of a human source.
 23. The method of claim 13, wherein said insulinsecreting cells are of a histocompatibility humanized animal source. 24.The method of claim 13, wherein said insulin secreting cells secretehuman insulin.
 25. The method of claim 13, wherein said insulinsecreting cells are autologous cells.
 26. The method of claim 13,wherein said cells are selected from the group consisting offibroblasts, epithelial cells and keratinocytes.
 27. A method ofinducing or accelerating a healing process of a skin wound, the methodcomprising the step of transforming cells of the skin wound to produceand secrete insulin, so as to induce or accelerate the healing processof the skin wound.
 28. The method of claim 27, wherein said wound isselected from the group consisting of an ulcer, a burn, a laceration anda surgical incision
 29. The method of claim 28, wherein said ulcer is adiabetic ulcer.
 30. The method of claim 27, wherein said cells aretransformed by a recombinant PDX1 gene and therefore said cells produceand secrete natural insulin.
 31. The method of claim 27, wherein saidcells are transformed by a cis-acting element sequence integratedupstream to an endogenous insulin gene of said cells and therefore saidcells produce and secrete natural insulin.
 32. The method of claim 27,wherein said cells are transformed by a recombinant insulin gene andtherefore said cells produce and secrete recombinant insulin.
 33. Amethod of inducing or accelerating a healing process of a skin wound,the method comprising the step of transforming cells of said skin woundto produce a protein kinase C, so as to induce or accelerate the healingprocess of the skin wound.
 34. The method of claim 33, wherein said skinwound is selected from the group consisting of an ulcer, a burn, alaceration and a surgical incision
 35. The method of claim 34, whereinsaid ulcer is a diabetic ulcer.
 36. The method of claim 33, wherein saidcells are transformed to produce a protein kinase C transcriptionactivator and therefore said cells produce natural protein kinase C. 37.The method of claim 33, wherein said cells are transformed by acis-acting element sequence integrated upstream to an endogenous proteinkinase C of said cells and therefore said cells produce natural proteinkinase C.
 38. The method of claim 33, wherein said cells are transformedby a recombinant protein kinase C gene and therefore said cells producerecombinant protein kinase C.
 39. The method of claim 33, wherein saidprotein kinase C is selected from the group consisting of PKC-β1,PKC-β2, PKC-γ, PKC-θ, PKC-λ, and PKC-ι.
 40. The method of claim 33,wherein said protein kinase C is selected from the group consisting ofPKC-α, PKC-δ, PKC-ε, PKC-η, and PKC-ζ.
 41. A pharmaceutical compositionfor inducing or accelerating a healing process of a skin wound, thepharmaceutical composition comprising, as an active ingredient, atherapeutically effective amount of insulin and at least one additionalagent acting in synergy with said insulin, and a pharmaceuticallyacceptable carrier being designed for topical application of thepharmaceutical composition.
 42. The pharmaceutical composition of claim41, wherein said at least one additional agent is a growth factor. 43.The pharmaceutical composition of claim 42, wherein said growth factoris a platelet-derived growth factor.
 44. The pharmaceutical compositionof claim 41, wherein said at least one additional agent is a PKC-αinhibitor.
 45. The pharmaceutical composition of claim 41, wherein saidinsulin is a recombinant.
 46. The pharmaceutical composition of claim41, wherein said insulin is of a natural source.
 47. The pharmaceuticalcomposition of claim 41, wherein said wound is selected from the groupconsisting of an ulcer, a burn, a laceration and a surgical incision.48. The pharmaceutical composition of claim 41, wherein said ulcer is adiabetic ulcer.
 49. The pharmaceutical composition of claim 41, whereinsaid insulin and at least one additional agent is contained in aformulation adapted for topical application.
 50. The pharmaceuticalcomposition of claim 49, wherein said formulation is selected from thegroup consisting of an aqueous solution, a gel, a cream, a paste, alotion, a spray, a suspension, a powder, a dispersion, a salve and anointment.
 51. The pharmaceutical composition of claim 50, wherein saidpharmaceutical composition includes a solid support.
 52. Apharmaceutical composition for inducing or accelerating a healingprocess of a skin wound, the pharmaceutical composition comprising, asan active ingredient, insulin secreting cells, and a pharmaceuticallyacceptable carrier being designed for topical application of thepharmaceutical composition.
 53. The pharmaceutical composition of claim52, wherein said wound is selected from the group consisting of anulcer, a burn, a laceration and a surgical incision.
 54. Thepharmaceutical composition of claim 53, wherein said ulcer is a diabeticulcer.
 55. The pharmaceutical composition of claim 52, wherein saidcells are transformed to produce and secrete insulin.
 56. Thepharmaceutical composition of claim 52, wherein said cells aretransformed by a recombinant PDX1 gene and therefore said cells produceand secrete natural insulin.
 57. The pharmaceutical composition of claim52, wherein said cells are transformed by a cis-acting element sequenceintegrated upstream to an endogenous insulin gene of said cells andtherefore said cells produce and secrete natural insulin.
 58. Thepharmaceutical composition of claim 52, wherein said cells aretransformed by a recombinant insulin gene and therefore said cellsproduce and secrete recombinant insulin.
 59. The pharmaceuticalcomposition of claim 52, wherein said insulin secreting cells arecapable of forming secretory granules.
 60. The pharmaceuticalcomposition of claim 52, wherein said insulin secreting cells areendocrine cells.
 61. The pharmaceutical composition of claim 52, whereinsaid insulin secreting cells are of a human source.
 62. Thepharmaceutical composition of claim 52, wherein said insulin secretingcells are of a histocompatibility humanized animal source.
 63. Thepharmaceutical composition of claim 52, wherein said insulin secretingcells secrete human insulin.
 64. The pharmaceutical composition of claim52, wherein said insulin secreting cells are autologous cells.
 65. Thepharmaceutical composition of claim 52, wherein said cells are selectedfrom the group consisting of fibroblasts, epithelial cells andkeratinocytes.
 66. A pharmaceutical composition for inducing oraccelerating a healing process of a skin wound, the pharmaceuticalcomposition comprising, as an active ingredient, a nucleic acidconstruct being designed for transforming cells of said skin wound toproduce and secrete insulin, and a pharmaceutically acceptable carrierbeing designed for topical application of the pharmaceuticalcomposition.
 67. The pharmaceutical composition of claim 66, whereinsaid wound is selected from the group consisting of an ulcer, a burn, alaceration and a surgical incision
 68. The pharmaceutical composition ofclaim 67, wherein said ulcer is a diabetic ulcer.
 69. The pharmaceuticalcomposition of claim 66, wherein said cells are transformed by arecombinant PDX1 gene and therefore said cells produce and secretenatural insulin.
 70. The pharmaceutical composition of claim 66, whereinsaid cells are transformed by a cis-acting element sequence integratedupstream to an endogenous insulin gene of said cells and therefore saidcells produce and secrete natural insulin.
 71. The pharmaceuticalcomposition of claim 66, wherein said cells are transformed by arecombinant insulin gene and therefore said cells produce and secreterecombinant insulin.
 72. A pharmaceutical composition for inducing oraccelerating a healing process of a skin wound, the pharmaceuticalcomposition comprising, as an active ingredient, a nucleic acidconstruct being designed for transforming cells of said skin wound toproduce a protein kinase C, and a pharmaceutically acceptable carrierbeing designed for topical application of the pharmaceuticalcomposition.
 73. The pharmaceutical composition of claim 72, whereinsaid skin wound is selected from the group consisting of an ulcer, aburn, a laceration and a surgical incision
 74. The pharmaceuticalcomposition of claim 73, wherein said ulcer is a diabetic ulcer.
 75. Thepharmaceutical composition of claim 72, wherein said cells aretransformed to produce a protein kinase C transcription activator andtherefore said cells produce natural protein kinase C.
 76. Thepharmaceutical composition of claim 72, wherein said cells aretransformed by a cis-acting element sequence integrated upstream to anendogenous protein kinase C of said cells and therefore said cellsproduce natural protein kinase C.
 77. The pharmaceutical composition ofclaim 72, wherein said cells are transformed by a recombinant proteinkinase C gene and therefore said cells produce recombinant proteinkinase C.
 78. The pharmaceutical composition of claim 72, wherein saidprotein kinase C is selected from the group consisting of PKC-β1,PKC-β2, PKC-γ, PKC-θ, PKC-λ, and PKC-ι.
 79. The pharmaceuticalcomposition of claim 72, wherein said protein kinase C is selected fromthe group consisting of PKC-α, PKC-δ, PKC-ε, PKC-η and PKC-ζ.
 80. Amethod of inducing or accelerating a healing process of a skin wound,the method comprising the step of administering to the skin wound atherapeutically effective amount of an agent for modulating PKCproduction and/or activation.
 81. A pharmaceutical composition forinducing or accelerating a healing process of a skin wound, thepharmaceutical composition comprising, as an active ingredient, atherapeutically effective amount of an agent for modulating PKCproduction and/or activation; and a pharmaceutically acceptable carrier.82. A method of inducing or accelerating a healing process of a skinwound, the method comprising the step of administering to the skin wounda therapeutically effective amount of a PKC activator, so as to induceor accelerate the healing process of the skin wound.
 83. Apharmaceutical composition of inducing or accelerating a healing processof a skin wound, the pharmaceutical composition comprising, as an activeingredient, a therapeutically effective amount of a PKC activator, so asto induce or accelerate the healing process of the skin wound, and anacceptable pharmaceutical carrier.
 84. A method of inducing oraccelerating ex-vivo propagation of skin cells, the method comprisingthe step of subjecting the skin cells to an effective amount of an agentfor modulating PKC production.
 85. A method of inducing or acceleratinga healing process of a skin wound, the method comprising the step ofadministering to the skin wound a single dose of a therapeuticallyeffective amount of insulin, thereby inducing or accelerating thehealing process of said skin wound.
 86. The method of claim 85, whereinsaid skin wound is selected from the group consisting of an ulcer, aburn, a laceration and a surgical incision.
 87. The method of claim 86,wherein said ulcer is a diabetic ulcer.
 88. The method of claim 85,wherein said insulin is recombinant.
 89. The method of claim 85, whereinsaid insulin is of a natural source.
 90. The method of claim 85, whereinsaid insulin is contained in a pharmaceutical composition adapted fortopical application.
 91. The method of claim 90, wherein saidpharmaceutical composition is selected from the group consisting of anaqueous solution, a gel, a cream, a paste, a lotion, a spray, asuspension, a powder, a dispersion, a salve and an ointment.
 92. Themethod of claim 90, wherein said pharmaceutical composition includes asolid support.
 93. A method of inducing or accelerating a healingprocess of an old skin wound, the method comprising the step ofadministering to the old skin wound a single dose of a therapeuticallyeffective amount of insulin, thereby inducing or accelerating thehealing process of the old skin wound.
 94. The method of claim 93,wherein said old skin wound is at least 2 days old.
 95. The method ofclaim 93, wherein said old skin wound is selected from the groupconsisting of an ulcer, a bum, a laceration and a surgical incision. 96.The method of claim 95, wherein said ulcer is a diabetic ulcer.
 97. Themethod of claim 93, wherein said insulin is recombinant.
 98. The methodof claim 93, wherein said insulin is of a natural source.
 99. The methodof claim 93, wherein said insulin is contained in a pharmaceuticalcomposition adapted for topical application.
 100. The method of claim99, wherein said pharmaceutical composition is selected from the groupconsisting of an aqueous solution, a gel, a cream, a paste, a lotion, aspray, a suspension, a powder, a dispersion, a salve and an ointment.101. The method of claim 99, wherein said pharmaceutical compositionincludes a solid support.
 102. A pharmaceutical composition for inducingor accelerating a healing process of a skin wound, the pharmaceuticalcomposition comprising, as an active ingredient, a single dose-unit ofinsulin selected capable of inducing or accelerating a healing processof the skin wound, and a pharmaceutically acceptable carrier beingdesigned for topical application of the pharmaceutical composition. 103.The pharmaceutical composition of claim 102, wherein said singledose-unit of insulin is 0.001 to 5 nM in 0.01-0.2 ml of saidpharmaceutical composition.
 104. The pharmaceutical composition of claim102, wherein said single dose of insulin is ranging from 0.01 to 0.5 nMin 0.01-0.2 ml of said pharmaceutical composition.
 105. Thepharmaceutical composition of claim 102, wherein said insulin is arecombinant.
 106. The pharmaceutical composition of claim 102, whereinsaid insulin is of a natural source.
 107. The pharmaceutical compositionof claim 102, wherein said skin wound is selected from the groupconsisting of an ulcer, a burn, a laceration and a surgical incision.108. The pharmaceutical composition of claim 102, wherein said ulcer isa diabetic ulcer.
 109. The pharmaceutical composition of claim 102,wherein said insulin is contained in a formulation adapted for topicalapplication.
 110. The pharmaceutical composition of claim 109, whereinsaid formulation is selected from the group consisting of an aqueoussolution, a gel, a cream, a paste, a lotion, a spray, a suspension, apowder, a dispersion, a salve and an ointment.
 111. The pharmaceuticalcomposition of claim 102, wherein said pharmaceutical compositionincludes a solid support.